Comparison of neuroglobin distribution and expression between the retina of the adult Bactrian camel, rabbits and sheep

Med.Weter. D0:dx.doi.org/10.21521/mw.6473 Praca oryginalna Original paper Comparison of neuroglobin distribution and expression between the retina of the adult Bactrian camel,rabbits and sheep1) XIAOHUA DU,JAMES BLACKAR MAWOLO*,XIA LIU* College of Veterinary Medicine,*College of Life Science and Technology, Gansu Agricultural University,Lanzhou City,Gansu Province,People's Republic of China Received 01.06.2020 Accepted 10.07.2020 Du X.,Mawolo J.B.,Liu X. Comparison of neuroglobin distribution and expression between the retina of the adult Bactrian camel, rabbits and sheep Summary Neuroglobin(Ngb)is a kind of protein largely expressed in the brain and retina of mammals.Numerous studies have reported on Ngb expression and distribution in mammals but none have compared the expression in the adult Bactrian camel,rabbits,and sheep.The study examined the distribution and expression of Ngb between the retina of adult Bactrian camel,rabbits,and sheep and provides detailed insight on the morphology of these mammals'retinae.The immunohistochemical staining procedures were performed to detect Ngb distribution and its expression in the retinae of the adult Bactrian camel,rabbits,and sheep.The results showed that strong positive Ngb expression was found in all layers of the Bactrian camel except the outer nuclear layer,while in the rabbit retina,the strong positive expression was observed in the cortex of the optic nerve fiber layer,the retina cells layer,the network layer,the photoreceptor inner segment,and the pigment,while weak positive expression was shown in the retina of the kernel layer,outside the outer nuclear layer of the retina and the light receptor section.In the adult sheep retina,Ngb was solely expressed in the nerve fiber layer,inner and outer plexiform layer,optic nerve,inner and outer limiting membrane,and photoreceptor inner segment,while weak positive expression was shown in the ganglion cell layer and inner nuclear layer.There exist no Ngb positive expression in the photoreceptor outer segment,the outer nuclear layer,and retinal pigment epithelium of the adult sheep retina.The study documented that Ngb may have a significant function in the maintenance of retinal oxygen homeostasis and participation in the repair of light damage.The study also provided detailed references for Ngb physiological function and its relationship to extreme environmental conditions. Keywords:Neuroglobin,retina,immunohistochemistry,Bactrian camel,rabbit and sheep Neuroglobin (Ngb)is a neuronal hemeprotein ofoxygen consumption(8).Scientists have studied the similar to myoglobin and hemoglobin and shares their expression of Ngb in the retina of rodents and humans capability for oxygen binding.Research has shown that (10,11)and reported significant references about these Ngb expression in the retina is higher than other neural animals.The expression level of Ngb in the retina is tissues,and its concentration is about 50-100 times much higher than in the brain tissue(12,13),suggest- (26).Yu et al.reported that in species with vascularized ing that there exists a close relationship between Ngb retina,oxygen is transported to the retina in combina- and retinal functions.Studies have shown that Ngb is tion with the choroidal vascular bed.which is located involved in the elimination of reactive oxygen species at the back of the retina and is the highest consumer of (ROS)regulation which may play an important role in oxygen(5).Another study confirmed that the ocular tis- oxygen homeostasis(14).Further research conducted sues in the retina are unique and have the highest rates by Ostoji et al.reported that the expression of Ngb was The research project was funded by the National Natural Science Foundation solely found in the ganglion cell layer,inner nuclear of China(Grant No.31760305),The Youth Foundation of Gansu Agricultural layer,inner and outer plexiform layers,and retinal pig- University (Grant No.GAU-QNDS-201501),and the National Natural Foun- dation of Gansu Province(Grant No.17JR5RA142).The funders had no role in ment epithelium but not in the glial,suggesting that study design,data collection,and analysis,decision to publish,or preparation Ngb could serve a neuroprotective role as scavengers of the manuscript. of reactive oxygen species and developing therapeutic

Med. Weter. 1 Praca oryginalna Original paper DOI: dx.doi.org/10.21521/mw.6473 Neuroglobin (Ngb) is a neuronal hemeprotein similar to myoglobin and hemoglobin and shares their capability for oxygen binding. Research has shown that Ngb expression in the retina is higher than other neural tissues, and its concentration is about 50-100 times (26). Yu et al. reported that in species with vascularized retina, oxygen is transported to the retina in combina￾tion with the choroidal vascular bed, which is located at the back of the retina and is the highest consumer of oxygen (5). Another study confirmed that the ocular tis￾sues in the retina are unique and have the highest rates of oxygen consumption (8). Scientists have studied the expression of Ngb in the retina of rodents and humans (10, 11) and reported significant references about these animals. The expression level of Ngb in the retina is much higher than in the brain tissue (12, 13), suggest￾ing that there exists a close relationship between Ngb and retinal functions. Studies have shown that Ngb is involved in the elimination of reactive oxygen species (ROS) regulation which may play an important role in oxygen homeostasis (14). Further research conducted by Ostoji et al. reported that the expression of Ngb was solely found in the ganglion cell layer, inner nuclear layer, inner and outer plexiform layers, and retinal pig￾ment epithelium but not in the glial, suggesting that Ngb could serve a neuroprotective role as scavengers of reactive oxygen species and developing therapeutic Comparison of neuroglobin distribution and expression between the retina of the adult Bactrian camel, rabbits and sheep1) XIAOHUA DU, JAMES BLACKAR MAWOLO*, XIA LIU* College of Veterinary Medicine, *College of Life Science and Technology, Gansu Agricultural University, Lanzhou City, Gansu Province, People’s Republic of China Received 01.06.2020 Accepted 10.07.2020 Du X., Mawolo J. B., Liu X. Comparison of neuroglobin distribution and expression between the retina of the adult Bactrian camel, rabbits and sheep Summary Neuroglobin (Ngb) is a kind of protein largely expressed in the brain and retina of mammals. Numerous studies have reported on Ngb expression and distribution in mammals but none have compared the expression in the adult Bactrian camel, rabbits, and sheep. The study examined the distribution and expression of Ngb between the retina of adult Bactrian camel, rabbits, and sheep and provides detailed insight on the morphology of these mammals’ retinae. The immunohistochemical staining procedures were performed to detect Ngb distribution and its expression in the retinae of the adult Bactrian camel, rabbits, and sheep. The results showed that strong positive Ngb expression was found in all layers of the Bactrian camel except the outer nuclear layer, while in the rabbit retina, the strong positive expression was observed in the cortex of the optic nerve fiber layer, the retina cells layer, the network layer, the photoreceptor inner segment, and the pigment, while weak positive expression was shown in the retina of the kernel layer, outside the outer nuclear layer of the retina and the light receptor section. In the adult sheep retina, Ngb was solely expressed in the nerve fiber layer, inner and outer plexiform layer, optic nerve, inner and outer limiting membrane, and photoreceptor inner segment, while weak positive expression was shown in the ganglion cell layer and inner nuclear layer. There exist no Ngb positive expression in the photoreceptor outer segment, the outer nuclear layer, and retinal pigment epithelium of the adult sheep retina. The study documented that Ngb may have a significant function in the maintenance of retinal oxygen homeostasis and participation in the repair of light damage. The study also provided detailed references for Ngb physiological function and its relationship to extreme environmental conditions. Keywords: Neuroglobin, retina, immunohistochemistry, Bactrian camel, rabbit and sheep 1) The research project was funded by the National Natural Science Foundation of China (Grant No. 31760305), The Youth Foundation of Gansu Agricultural University (Grant No. GAU-QNDS-201501), and the National Natural Foun￾dation of Gansu Province (Grant No. 17JR5RA142). The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript

Med.Weter. strategies for the treatment of hypoxia-related ocular clonal antibody(Bs-1858r,Beijing,China)for one night at diseases (15,29).Despite all these results,there are 4C.The sections were rinsed in PBS and incubated at room no references for Ngb expression in the retina of the temperature for 80 minutes.The sections were re-washed Bactrian camel,rabbits,and sheep.The Bactrian camel and embedded in Trypsin(T-815025,Beijing,China).After (Bactrian camel)is a domestic animal that lives in that.the sections (70%alcohol (night).80%alcohol (5 h). desert and semi-desert areas and is highly adaptable 90%alcohol (3 h).95%alcohol (2 h).and n-butanol (night) to the extreme natural environment of drought and converted to n-butanol,(5 h)were prepared and embedded heat while adult rabbits are small mammals in the into paraffin wax.tissue sections were cut and examined family Leporidae and need a regulated environment to under an optical microscope (Olympus BX51). protect them against heat exhaustion or hypothermia. Reagents and instruments.The Trypsin(T-815025), Sheep are ruminant(cud-chewing)mammals of the Phosphate-buffered saline (PBS,Zli-9062)and rabbit genus Ovis and live throughout the world,especially anti-rat polyclonal antibody (Bs-1858r)were purchased in mountainous areas.The current research examined from Beijing Bio-Dee Biotechnology Co.Ltd.while the Ngb expression in the retina of healthy adult Bactrian immunohistochemical staining kit (sp-9001)and DAB color developing kit (Zli-9018)were ordered at Beijing camels,rabbits,and sheep by employing immuno- Zhongshan Jingiao Biotechnology Co.Ltd and Formalde- histochemical staining procedures.The research also hyde and n-butanol solution,analytically pure came from documented a theoretical foundation for understanding Tianjin Baishi Chemical Co.LTD.Glacial acetic acid and the repair function of Ngb in the retina injury of mam- alcohol solution:analysis-pure were bought from Tianjin mals under extreme natural environment and supplied Fuyu Fine Chemical Co Ltd and the normal sheep serum morphological data. (ab7489)came from North America.The following medical Material and methods instruments were purchased and employed during the sam- pling procedures:conventional surgical instruments which Animals and setting.The procedures used in this study include:scalpel and dissecting scissors.biological tissue were reviewed and approved by the Animal Ethics and Wel- embedding KD-BM machine,Bio-texturing and spread- fare Committee of Approval Number:Gansu Agricultural ing machine(K-P),and Biological tissue baking machine University (GAU-AEW-2019-0026). (KD-H)were purchased from Zhejiang Medical Getidy A total of three (3)healthy adult camels(7 years),five Medical Instruments Co.Zhejiang,Jinhua.The Constant (5)adult rabbits(6 years),and five(5)healthy adult sheep temperature incubator DHP-9082 was ordered at Shanghai (6 years)were involved in the study.The healthy adult Tajilin Industrial Co.Ltd,while the Semi-self-actuating camels were purchased from the Zhangye prefecture-level screw-cutting machine with a precision roller(Rm-2235) areas in Wuwei,Gansu province,China while the rabbits was bought from Leica,Germany,and Optical microscope and sheep used in this study were taken from Lanzhou (13395H2X)was purchased at Leica.Germany. city in Gansu Province.China were tested,and showed no Immuno-histochemical coloration.Streptavidin Per- sign of any disease.Specimens were collected in a morn- oxidase (SP)method was used for immunohistochemical ing and evening hour during July and collection was done staining while PBS was utilized as a negative control instead within one week.Animals were retrieved one after another of the primary antibody.Sheep serum was added to block from their housing area and taken to the slaughterhouse. the non-specific reactivity in each section and then observed Zhangye prefecture is located in central Gansu along the under the optical microscope (Olympus 13395H2X)to Hexi Corridor,occupying 42,000 km2(16,000 sq mi).It control color rendering.The color rendering period for takes up the entire breadth of the province,running from Inner Mongolia on the north to Qinghai on the south,but each section was consistent.After the reaction stopped by distilled water,the slices were re-dyed,dehydrated,and its urban core is at Ganzhou in the oasis formed by the Ruo transparent by hematoxylin and sealed.A Nikon Eclipse 80i, or Hei River and the Lanzhou city is the capital and larg- micro camera system was employed to capture specimen est city of Gansu Province and is noted for its yellow river images with consent shooting parameters and the Image- and situated in the temperate zone with a semi-arid climate during hot summer and extremely cold and dry winter.The Pro Plus 6.0 was introduced to acquire,count,measure,and average monthly temperature ranges from-5.3C(22.5F) classify optical density of each retina layer. Statistical analysis.Data analysis was performed using in January to 22.4C(72.3F)in July. Samples extraction and preparation.Before been Statistical Package Social Sciences(SPSS)1.0(1983)and 19.0(2009)to conduct one-way ANOVA,multiple com- sacrificed.all animals were fed and rested for a while.The animals were sacrificed,the cornea was open with a sharp parisons (Tamhane,ST2 method),and the Independent blade and the eyeballs were quickly removed and placed in Sample T-test was employed to evaluate the significant Davidson's fixation solution(95%alcohol,37%strong for- level.P0.05),and positive (+control or retinae were placed on disinfected glass slides,and normal expression if there was any significant difference. sheep serum(ab7489,North America)was added to block Results and discussion the non-specific binding sides at room temperature for an hour in Phosphate-buffered saline(PBS,Beijing,China). Adult Bactrian camel.The reports from HE stain- These sections were incubated with a rabbit anti-Ngb poly- ing showed that the Bactrian camel structure contains

2 Med. Weter. strategies for the treatment of hypoxia-related ocular diseases (15, 29). Despite all these results, there are no references for Ngb expression in the retina of the Bactrian camel, rabbits, and sheep. The Bactrian camel (Bactrian camel) is a domestic animal that lives in desert and semi-desert areas and is highly adaptable to the extreme natural environment of drought and heat while adult rabbits are small mammals in the family Leporidae and need a regulated environment to protect them against heat exhaustion or hypothermia. Sheep are ruminant (cud-chewing) mammals of the genus Ovis and live throughout the world, especially in mountainous areas. The current research examined Ngb expression in the retina of healthy adult Bactrian camels, rabbits, and sheep by employing immuno￾histochemical staining procedures. The research also documented a theoretical foundation for understanding the repair function of Ngb in the retina injury of mam￾mals under extreme natural environment and supplied morphological data. Material and methods Animals and setting. The procedures used in this study were reviewed and approved by the Animal Ethics and Wel￾fare Committee of Approval Number: Gansu Agricultural University (GAU-AEW-2019-0026). A total of three (3) healthy adult camels (7 years), five (5) adult rabbits (6 years), and five (5) healthy adult sheep (6 years) were involved in the study. The healthy adult camels were purchased from the Zhangye prefecture-level areas in Wuwei, Gansu province, China while the rabbits and sheep used in this study were taken from Lanzhou city in Gansu Province, China were tested, and showed no sign of any disease. Specimens were collected in a morn￾ing and evening hour during July and collection was done within one week. Animals were retrieved one after another from their housing area and taken to the slaughterhouse. Zhangye prefecture is located in central Gansu along the Hexi Corridor, occupying 42,000 km2 (16,000 sq mi). It takes up the entire breadth of the province, running from Inner Mongolia on the north to Qinghai on the south, but its urban core is at Ganzhou in the oasis formed by the Ruo or Hei River and the Lanzhou city is the capital and larg￾est city of Gansu Province and is noted for its yellow river and situated in the temperate zone with a semi-arid climate during hot summer and extremely cold and dry winter. The average monthly temperature ranges from –5.3°C (22.5°F) in January to 22.4°C (72.3°F) in July. Samples extraction and preparation. Before been sacrificed, all animals were fed and rested for a while. The animals were sacrificed, the cornea was open with a sharp blade and the eyeballs were quickly removed and placed in Davidson’s fixation solution (95% alcohol, 37% strong for￾malin, glacial acetic acid, distilled water ratio: 3 : 2 : 1 : 2) for 24 hours and stored at 4°C. Later, the perfusion-fixed retinae were placed on disinfected glass slides, and normal sheep serum (ab7489, North America) was added to block the non-specific binding sides at room temperature for an hour in Phosphate-buffered saline (PBS, Beijing, China). These sections were incubated with a rabbit anti-Ngb poly￾clonal antibody (Bs-1858r, Beijing, China) for one night at 4°C. The sections were rinsed in PBS and incubated at room temperature for 80 minutes. The sections were re-washed and embedded in Trypsin (T-815025, Beijing, China). After that, the sections (70% alcohol (night), 80% alcohol (5 h), 90% alcohol (3 h), 95% alcohol (2 h), and n-butanol (night) converted to n-butanol, (5 h) were prepared and embedded into paraffin wax, tissue sections were cut and examined under an optical microscope (Olympus BX51). Reagents and instruments. The Trypsin (T-815025), Phosphate-buffered saline (PBS, Zli-9062) and rabbit anti-rat polyclonal antibody (Bs-1858r) were purchased from Beijing Bio-Dee Biotechnology Co. Ltd. while the immunohistochemical staining kit (sp-9001) and DAB color developing kit (Zli-9018) were ordered at Beijing Zhongshan Jinqiao Biotechnology Co. Ltd and Formalde￾hyde and n-butanol solution, analytically pure came from Tianjin Baishi Chemical Co. LTD. Glacial acetic acid and alcohol solution: analysis-pure were bought from Tianjin Fuyu Fine Chemical Co Ltd and the normal sheep serum (ab7489) came from North America. The following medical instruments were purchased and employed during the sam￾pling procedures: conventional surgical instruments which include: scalpel and dissecting scissors, biological tissue embedding KD-BM machine, Bio-texturing and spread￾ing machine (K-P), and Biological tissue baking machine (KD-H) were purchased from Zhejiang Medical Getidy Medical Instruments Co. Zhejiang, Jinhua. The Constant temperature incubator DHP-9082 was ordered at Shanghai Tajilin Industrial Co. Ltd, while the Semi-self-actuating screw-cutting machine with a precision roller (Rm-2235) was bought from Leica, Germany, and Optical microscope (13395H2X) was purchased at Leica, Germany. Immuno-histochemical coloration. Streptavidin Per￾oxidase (SP) method was used for immunohistochemical staining while PBS was utilized as a negative control instead of the primary antibody. Sheep serum was added to block the non-specific reactivity in each section and then observed under the optical microscope (Olympus 13395H2X) to control color rendering. The color rendering period for each section was consistent. After the reaction stopped by distilled water, the slices were re-dyed, dehydrated, and transparent by hematoxylin and sealed. A Nikon Eclipse 80i, micro camera system was employed to capture specimen images with consent shooting parameters and the Image￾Pro Plus 6.0 was introduced to acquire, count, measure, and classify optical density of each retina layer. Statistical analysis. Data analysis was performed using Statistical Package Social Sciences (SPSS) 1.0 (1983) and 19.0 (2009) to conduct one-way ANOVA, multiple com￾parisons (Tamhane, ST2 method), and the Independent Sample T-test was employed to evaluate the significant level. P 0.05), and positive (+) control or expression if there was any significant difference. Results and discussion Adult Bactrian camel. The reports from HE stain￾ing showed that the Bactrian camel structure contains

Med.Weter. Fig.1.Distribution of neuroglobin in the retina of the adult Bactrian camel 10 layers with normal cell stratification,which are ing a dark cell layer(Fig.1C).The layers of cells form similar to other mammals.The characteristics of each a complex neural network in the horizontal and vertical layer are described as follows:The inner boundary directions to complete the processing of information membrane contains the miller and glial cells,accom- The immuno-histochemical results showed that Ngb panied by vitreous fibers covering the whole retinal expression was detected in all layers except the outer surface.It is referred to as the innermost layer of the nuclear layer of Bactrian camel retina(Fig.1A and 1B). homogeneous film.While the optic nerve fiber layer, At the cellular level,Ngb is expressed intensively in the when examined under the light microscope,is a criss- cytoplasm of pigment epithelial cells,photoreceptor crossed and porous network structure which composed cells,bipolar cells,and nodal cells.No positive expres- of ganglion cell axons and has small blood vessels that sion was found in the negative control group (Fig.1D) were observed.The ganglion cell layer is composed of Moreover,positive Ngb distribution was observed in clearly arranged ganglion cells and nucleus of differ- the optic nerve of the Bactrian camel(Fig.1E and 1F). ent sizes and the inner reticular layer:a dense reticular The reported results of Intel's integrated performance structure formed by the protuberance of nodal cells and primitives (IPP)and statistical analysis showed that bipolar cells;on the other hand,the core layer is de- the MD value of the outer nuclear layer was 0.0334 scribed as the loose nuclear layer composed of bipolar +0.0144,explaining no significant existence in the and horizontal cells which can be observed under the negative (-)control group(P>0.05).The MD values light microscope.The outer reticular layer is a dense reticular structure composed of Tab.1.The comparison of the Ngb expression intensity in adult Bactrian dendrites of horizontal and bipolar cells and camel axons of photoreceptor cells and the outer Regions Main Optical Density The expression Density nuclear layer contains a dense nuclear layer Nerve fiber layer(NFL) 0.1762±0.0707*4 ++ composed of photoreceptor cells with a tight Ganglion Cell layer(GCL) 0.1611±0.0491**a ++ arrangement and clear nucleus which can be examined under the light microscope.The Inner plexiform layer(IPL) 0.1424±0.0464** outer membrane is a thin and clear membrane Inner nuclear layer(INL) 0.0794±0.0238**4 + structure composed of muller cells and the Outer plexiform layer(OPL) 0.1407±0.0453**4 ++ rod-cone layer is a region composed of rod Outer nuclear layer(ONL) 0.0334±0.0144 and cone cells,which can be divided into two Photoreceptor inner segments(IS) 0.1756±0.0502**4 ++ parts under the light microscope:internal and Photoreceptor outer Segments(OS) 0.0749±0.0229 external segments of photoreceptors.Another region is the pigment super cortex which is Pigmented epithelial layer(PEL) 0.1985±0.0803*4 ++ located in the outermost part of the retina and Explanations:*positive and negative control group,P>0.05;**positive and consists of pigment cells and flat cells form- negative control group,P<0.05;positive control group,P<0.05

Med. Weter. 3 10 layers with normal cell stratification, which are similar to other mammals. The characteristics of each layer are described as follows: The inner boundary membrane contains the miller and glial cells, accom￾panied by vitreous fibers covering the whole retinal surface. It is referred to as the innermost layer of the homogeneous film. While the optic nerve fiber layer, when examined under the light microscope, is a criss￾crossed and porous network structure which composed of ganglion cell axons and has small blood vessels that were observed. The ganglion cell layer is composed of clearly arranged ganglion cells and nucleus of differ￾ent sizes and the inner reticular layer: a dense reticular structure formed by the protuberance of nodal cells and bipolar cells; on the other hand, the core layer is de￾scribed as the loose nuclear layer composed of bipolar and horizontal cells which can be observed under the light microscope. The outer reticular layer is a dense reticular structure composed of dendrites of horizontal and bipolar cells and axons of photoreceptor cells and the outer nuclear layer contains a dense nuclear layer composed of photoreceptor cells with a tight arrangement and clear nucleus which can be examined under the light microscope. The outer membrane is a thin and clear membrane structure composed of muller cells and the rod-cone layer is a region composed of rod and cone cells, which can be divided into two parts under the light microscope: internal and external segments of photoreceptors. Another region is the pigment super cortex which is located in the outermost part of the retina and consists of pigment cells and flat cells form￾ing a dark cell layer (Fig. 1C). The layers of cells form a complex neural network in the horizontal and vertical directions to complete the processing of information. The immuno-histochemical results showed that Ngb expression was detected in all layers except the outer nuclear layer of Bactrian camel retina (Fig. 1A and 1B). At the cellular level, Ngb is expressed intensively in the cytoplasm of pigment epithelial cells, photoreceptor cells, bipolar cells, and nodal cells. No positive expres￾sion was found in the negative control group (Fig. 1D). Moreover, positive Ngb distribution was observed in the optic nerve of the Bactrian camel (Fig. 1E and 1F). The reported results of Intel’s integrated performance primitives (IPP) and statistical analysis showed that the MD value of the outer nuclear layer was 0.0334 ± 0.0144, explaining no significant existence in the negative (–) control group (P > 0.05). The MD values Tab. 1. The comparison of the Ngb expression intensity in adult Bactrian camel Regions Main Optical Density The expression Density Nerve fiber layer (NFL) 0.1762 ± 0.0707**▲ ++ Ganglion Cell layer (GCL) 0.1611 ± 0.0491**▲ ++ Inner plexiform layer (IPL) 0.1424 ± 0.0464**▲ ++ Inner nuclear layer (INL) 0.0794 ± 0.0238**▲ + Outer plexiform layer (OPL) 0.1407 ± 0.0453**▲ ++ Outer nuclear layer (ONL) 0.0334 ± 0.0144* – Photoreceptor inner segments (IS) 0.1756 ± 0.0502**▲ ++ Photoreceptor outer Segments (OS) 0.0749 ± 0.0229* + Pigmented epithelial layer (PEL) 0.1985 ± 0.0803**▲ ++ Explanations: * positive and negative control group, P > 0.05; ** positive and negative control group, P < 0.05; ▲ positive control group, P < 0.05 Fig. 1. Distribution of neuroglobin in the retina of the adult Bactrian camel

Med.Weter. of each region were significantly different from the axon of ganglion cells and the positive surface of negative contrast(P<0.05),and were determined to be the nodule cell layer was considered as the cell body a weakly positive (+)expression.Similarly,the regions of the nodule cell with different sizes and outlines. with MD values were higher than the kernel layer,and Immunohistochemical results showed that Ngb ex- the significant difference(P<0.05)was considered as pression was located in the cytoplasm,and no positive a strongly positive (++)expression,which was con- staining was observed in the nucleus.The Ngb positive sistent with the observation results under the optical expression of the inner reticular layer was distributed microscope (Tab.1). in the axon of the bipolar cell and dendrite of the node Adult rabbits.In the retina of adult rabbits,the NGB cell,while in the outer reticular layer it was found in the immuno-positive reaction was mainly distributed in axons of rods and cones,the dendrites of bipolar cells the optic nerve fiber layer,ganglion cell layer,inner and the protuberances of horizontal cells.The positive reticular layer,outer reticular layer,the inner segment expression of Ngb in the inner layer was the cell body of photoreceptor and pigment super-cortex,inner of bipolar cells,in which the cytoplasm was strongly boundary layer,outer membrane and inner layer(Fig.2).The Ngb positive expression found in the fibril was mainly located in the axon of ganglion cells while a similar expres- sion in the nodal cell layer was observed in the body of nodal cells with different sizes and specific lining.Immuno-histochemical results showed that NGB was localized in the cytoplasm,and no positive staining was observed in the nucleus.Ngb positive expression in the inner reticular layer was mainly the axons of bipolar cells and the dendrites of ganglion cells.The expression of Ngb in the outer reticular layer is the axons of rods and cones,the dendrites of bipolar cells,and the protuberances of horizontal cells.The pigment epithelial layer contains a positive expression of Ngb and it is located in the pigment epithelial cells.The positive expression of the inner layer was the cell body of bipolar cells,in which the cytoplasm was strongly colored.The cytotypes of Ngb positive phenotype are mainly photoreceptor cells,bipolar cells,and retinal ganglion cells, which are consistent with the third-order reti- nal neurons.No Ngb positive expression was found in the outer nuclear layer and the outer segment of the photoreceptor in the retina of adult rabbits.There were no positive signs in the negative control group(Fig.2 v).Besides. Fig.2.Distribution of neuroglobin in the retina of the adult rabbit immunization results showed that positive expression of NGB was observed in the optic Tab.2.The comparison of the Ngb expression intensity in the adult rabbit nerve of adult rabbits (Fig.2 iii,iv)and the Regions Main Optical Density The expression Density statistical analysis are shown in Tab.2. Nerve fiber layer(NFL) 0.151±0.050**4 x Adult sheep.The retina is the innermost Ganglion Cell layer(GCL) 0.173±0.040**4 x layer of the mammalian eye which contains Inner plexiform layer(IPL) 0.140±0.019*4 ten layers from inner to the outer one(Fig.3 米x i-vi).In the retina of adult sheep,the Ngb Inner nuclear layer(INL) 0.091±0.031◆+ + was mainly distributed in the optic nerve Outer plexiform layer(OPL) 0.123±0.017**“ x fiber layer,ganglion cell layer,inner and Outer nuclear layer(ONL) 0.020±0.013* outer reticular layer,the inner segment ofthe Photoreceptor inner segments(IS) 0.143±0.038**4 photoreceptor,while the positive expression Photoreceptor outer Segments(OS) 0.033±0.014 was found in the inner and outer membranes Pigmented epithelial layer(PEL) 0.170±0.040**1 (Fig.3).The positive expression of the optic nerve fiber layer was mainly located in the Explanations:as in Tab.1

4 Med. Weter. of each region were significantly different from the negative contrast (P < 0.05), and were determined to be a weakly positive (+) expression. Similarly, the regions with MD values were higher than the kernel layer, and the significant difference (P < 0.05) was considered as a strongly positive (++) expression, which was con￾sistent with the observation results under the optical microscope (Tab. 1). Adult rabbits. In the retina of adult rabbits, the NGB immuno-positive reaction was mainly distributed in the optic nerve fiber layer, ganglion cell layer, inner reticular layer, outer reticular layer, the inner segment of photoreceptor and pigment super-cortex, inner boundary layer, outer membrane and inner layer (Fig. 2). The Ngb positive expression found in the fibril was mainly located in the axon of ganglion cells while a similar expres￾sion in the nodal cell layer was observed in the body of nodal cells with different sizes and specific lining. Immuno-histochemical results showed that NGB was localized in the cytoplasm, and no positive staining was observed in the nucleus. Ngb positive expression in the inner reticular layer was mainly the axons of bipolar cells and the dendrites of ganglion cells. The expression of Ngb in the outer reticular layer is the axons of rods and cones, the dendrites of bipolar cells, and the protuberances of horizontal cells. The pigment epithelial layer contains a positive expression of Ngb and it is located in the pigment epithelial cells. The positive expression of the inner layer was the cell body of bipolar cells, in which the cytoplasm was strongly colored. The cytotypes of Ngb positive phenotype are mainly photoreceptor cells, bipolar cells, and retinal ganglion cells, which are consistent with the third-order reti￾nal neurons. No Ngb positive expression was found in the outer nuclear layer and the outer segment of the photoreceptor in the retina of adult rabbits. There were no positive signs in the negative control group (Fig. 2 v). Besides, immunization results showed that positive expression of NGB was observed in the optic nerve of adult rabbits (Fig. 2 iii, iv) and the statistical analysis are shown in Tab. 2. Adult sheep. The retina is the innermost layer of the mammalian eye which contains ten layers from inner to the outer one (Fig. 3 i-vi). In the retina of adult sheep, the Ngb was mainly distributed in the optic nerve fiber layer, ganglion cell layer, inner and outer reticular layer, the inner segment of the photoreceptor, while the positive expression was found in the inner and outer membranes (Fig. 3). The positive expression of the optic nerve fiber layer was mainly located in the axon of ganglion cells and the positive surface of the nodule cell layer was considered as the cell body of the nodule cell with different sizes and outlines. Immunohistochemical results showed that Ngb ex￾pression was located in the cytoplasm, and no positive staining was observed in the nucleus. The Ngb positive expression of the inner reticular layer was distributed in the axon of the bipolar cell and dendrite of the node cell, while in the outer reticular layer it was found in the axons of rods and cones, the dendrites of bipolar cells and the protuberances of horizontal cells. The positive expression of Ngb in the inner layer was the cell body of bipolar cells, in which the cytoplasm was strongly Tab. 2. The comparison of the Ngb expression intensity in the adult rabbit Regions Main Optical Density The expression Density Nerve fiber layer (NFL) 0.151 ± 0.050**▲ ++ Ganglion Cell layer (GCL) 0.173 ± 0.040**▲ ++ Inner plexiform layer (IPL) 0.140 ± 0.019**▲ ++ Inner nuclear layer (INL) 0.091 ± 0.031** + Outer plexiform layer (OPL) 0.123 ± 0.017**▲ ++ Outer nuclear layer (ONL) 0.020 ± 0.013* – Photoreceptor inner segments (IS) 0.143 ± 0.038**▲ ++ Photoreceptor outer Segments (OS) 0.033 ± 0.014* – Pigmented epithelial layer (PEL) 0.170 ± 0.040**▲ ++ Explanations: as in Tab. 1. Fig. 2. Distribution of neuroglobin in the retina of the adult rabbit

Med.Weter. 100m 100m 6 Fig.3.Distribution of neuroglobin in the retina of adult sheep colored.The main positive expression of cell Tab.3.The comparison of the Ngb expression intensity in sheep types was the photoreceptor cells,bipolar Regions Main Optical Density The expression Density cells,and retinal ganglion cells,which were Nerve fiber layer(NFL) 0.152±0.011*4 + consistent with the third-order neurons in the Ganglion Cell layer(GCL) 0.110±0.007*4 optic network membrane.No expression of Ngb exists in the outer nuclear layer of adult Inner plexiform layer(IPL) 0.121±0.011*4 sheep retina,the outer segment of the photo- Inner nuclear layer(INL) 0.078±0.2084 receptor,and the upper layer of chromatin. Outer plexiform layer(OPL) 0.015±0.001**4 + The Image-Pro Plus 6.0 semi-quantitative Outer nuclear layer(ONL) 0.013±0.001*+ + analysis software reported that the positive control group of sheep retinal optic nerve Photoreceptor inner segments(IS) 0.085±0.012*4 艹 fiber layer,thin cell layer,inner plexiform Photoreceptor outer Segments(OS) 0.016±0.004*+ layer,kernel layer,outer plexiform layer,the Pigmented epithelial layer(PEL) 0.018±0.012**4 + outer nuclear layer,photoreceptor inner seg- Explanations:as in Tab.1. ment,and pigments on the cortex photorecep- tor outer segments of Median Derivative(MD)values Adult Bactrian.The reported results in the current are mentioned(Tab.3).The T-test results read that the study showed that Ngb is positively expressed in all outer nuclear layer of the positive control group and layers of the Bactrian camel retina except the outer pigments on the cortex photoreceptor outer segments retinal nucleus,which is consistent with the results of showed no statistical significance as compared with previous studies on the adult rat,sheep and human(2, the negative(-)control group (P>0.0).The positive 9,16,17).Different research viewpoints are held on control group MD value of the optic nerve fiber layer, the distribution of NGB in the upper chromatin cor- inner plexiform layer,network layer,and photorecep- tex.Bentmann et al.(3)found that there was no NGB tor inner segment were higher than the ganglion cell positive substance in the pigment super cortex of rats layer and kernel layer(P<0.05),indicating that the and mice.However,Ostojic et al.(18)found that there optic nerve fiber layer and other layers were strongly was NGB positive expression in the pigment super positive(++).The ganglion and kernel cell layers were cortex of humans and dogs.Yang et al.(28)studies weakly positive (+)which was consistent with the reported that there was NGB distribution observed in visual observation of positive expression under a light the pigment super cortex of rabbits,but results on the microscope.Ngb was also positively expressed in the sheep did not find Ngb expression in the super cortex optic nerve of sheep(Fig.3i).Immunohistochemical pigment.Similarly to these reported results,the cur- results showed that except for the positive expression rent study also found Ngb positive distribution in the in the sclera sieve plate,positive expression of Ngb was retinal pigment of the Bactrian camel's super cortex. found in nerve fibers,glial cells,and central vascular These differences between previous results may be wall of the retina(Fig.3 iv,v). caused by different animal species,different living

Med. Weter. 5 colored. The main positive expression of cell types was the photoreceptor cells, bipolar cells, and retinal ganglion cells, which were consistent with the third-order neurons in the optic network membrane. No expression of Ngb exists in the outer nuclear layer of adult sheep retina, the outer segment of the photo￾receptor, and the upper layer of chromatin. The Image-Pro Plus 6.0 semi-quantitative analysis software reported that the positive control group of sheep retinal optic nerve fiber layer, thin cell layer, inner plexiform layer, kernel layer, outer plexiform layer, the outer nuclear layer, photoreceptor inner seg￾ment, and pigments on the cortex photorecep￾tor outer segments of Median Derivative (MD) values are mentioned (Tab. 3). The T-test results read that the outer nuclear layer of the positive control group and pigments on the cortex photoreceptor outer segments showed no statistical significance as compared with the negative (–) control group (P > 0.0). The positive control group MD value of the optic nerve fiber layer, inner plexiform layer, network layer, and photorecep￾tor inner segment were higher than the ganglion cell layer and kernel layer (P < 0.05), indicating that the optic nerve fiber layer and other layers were strongly positive (++). The ganglion and kernel cell layers were weakly positive (+), which was consistent with the visual observation of positive expression under a light microscope. Ngb was also positively expressed in the optic nerve of sheep (Fig. 3i). Immunohistochemical results showed that except for the positive expression in the sclera sieve plate, positive expression of Ngb was found in nerve fibers, glial cells, and central vascular wall of the retina (Fig. 3 iv, v). Adult Bactrian. The reported results in the current study showed that Ngb is positively expressed in all layers of the Bactrian camel retina except the outer retinal nucleus, which is consistent with the results of previous studies on the adult rat, sheep and human (2, 9, 16, 17). Different research viewpoints are held on the distribution of NGB in the upper chromatin cor￾tex. Bentmann et al. (3) found that there was no NGB positive substance in the pigment super cortex of rats and mice. However, Ostojic et al. (18) found that there was NGB positive expression in the pigment super cortex of humans and dogs. Yang et al. (28) studies reported that there was NGB distribution observed in the pigment super cortex of rabbits, but results on the sheep did not find Ngb expression in the super cortex pigment. Similarly to these reported results, the cur￾rent study also found Ngb positive distribution in the retinal pigment of the Bactrian camel’s super cortex. These differences between previous results may be caused by different animal species, different living Tab. 3. The comparison of the Ngb expression intensity in sheep Regions Main Optical Density The expression Density Nerve fiber layer (NFL) 0.152 ± 0.011**▲ ++ Ganglion Cell layer (GCL) 0.110 ± 0.007**▲ ++ Inner plexiform layer (IPL) 0.121 ± 0.011**▲ ++ Inner nuclear layer (INL) 0.078 ± 0.208▲ – Outer plexiform layer (OPL) 0.015 ± 0.001**▲ ++ Outer nuclear layer (ONL) 0.013 ± 0.001** ++ Photoreceptor inner segments (IS) 0.085 ± 0.012**▲ ++ Photoreceptor outer Segments (OS) 0.016 ± 0.004** ++ Pigmented epithelial layer (PEL) 0.018 ± 0.012**▲ ++ Explanations: as in Tab. 1. Fig. 3. Distribution of neuroglobin in the retina of adult sheep

Med.Weter. environments,different specificity of antibody qual- when Ngb protein expression increased or decreased, ity employed,and immunological detection.These it was accompanied by a corresponding decrease or changes lead to the different expression and localiza- increase in H,O,content,indicating that Ngb was tion of neuroglobin which are suggested to influence a ROS scavenger.On the other hand,Nayak et al.(16) the adaptive potential of an organism(1).The pigment found that Ngb produced by cultured nerve cells in cortex is located in the outermost layer of the retina, vitro can reduce ROS levels,indicating that Ngb plays and its main function is to supply oxygen and nutri- a certain role in ROS elimination.Similarly,the cur- ents to the outer retina and absorb light scattering. rent results have shown that Ngb positive expression Therefore,the pigment super cortex is vulnerable to of different intensity was found in the rods and cones the photochemical damage of visible light,which leads and the super cortex pigment,indicating that Ngb may to the accumulation ofreactive oxygen species(ROS), have an important repairing effect on retinal damage and then produces oxidative stress reaction,resulting caused by light.However,further research is needed in cell apoptosis.However,Ngb can remove excessive to confirm this report.The main function of the retina ROS,maintain the oxygen balance of tissue cells,and is visual conduction in which photoreceptors convert protect cells from light damage,which may also be one the received photochemical signals into electrical of the reasons for the different Ngb expression.Other signals,and can be transmitted to the central nervous results confirmed that oxygen affinity and enhanced system through various neurons in the optic network auto-oxidation question the role in oxygen delivery membrane forming vision.The current study reported and proposed that the neuroprotective effect might be that Ngb was expressed in the cytoplasm,rods,cones, due to radical scavenging or activation of protection and other cells of the retinal microcytes and bipolar mechanisms (4). cells.However,other studies have shown (22)that Researchers are still debating the exact location of there are a large number of synaptic connections of Ngb.Ran et al.(19)found that there were strong Ngb adjacent binary neurons between ganglion cells,bi- mRNA hybridization signals in the inner and outer polar cells and rod-cone cells to transmit information, nuclear layers of rats.However.the study of Xu et al. which is one of the regions with the highest oxygen (25)showed that Ngb was not expressed in the outer consumption in the retina and also the region with nuclear layer.The results of this current study show concentrated mitochondria,so it is very sensitive to that Ngb is not distributed in the outer nuclear layer, oxidative damage caused by ROS.The high expression which is consistent with Xu's report.Some studies of Ngb as a ROS scavenger in mitochondria suggests documented that there might be certain differences in that Ngb is maintaining a high oxygen consumption the spatial distribution of Ngb mRNA and protein in the group that has more complex physiological functions retina.which makes the retinal structure of mammals in tissue metabolism.Ngb is expressed with different to be partitioned.Ngb mRNA is mainly distributed in intensity in each layer of the retina,and its expression the cell body,while Ngb is distributed in the protru- level is positively correlated with the demand for tis- sion,suggesting that the Ngb protein has a transport sue cells for oxygen.Moreover,Ngb is an endogenous process after translation.In the present research,the neuroprotective agent,and the expression level sig- positive distribution of Ngb in the outer segment of the nificantly increased due to hypoxia and ischemia of photoreceptor is rarely reported.The current results tissue cells(21).Roberts et al.(20)who employed the revealed the presence of Ngb in the outer segment of method of constructing a mathematical model showed the photoreceptor.This result may be related to the that the effect of Ngb is not in the oxygen storage, different animal species or may be closely related to but the oxygen may play a more important role in the the function of the outer segment of the photorecep- models to predict photoreceptors in the retina anoxic tor during the visual formation of the Bactrian camel area in the segment and inner plexiform layer which under extreme environmental conditions.Whether is involved in the functioning of high expression of Ngb is related to the adaptability of the environment Ngb area.The expression of Ngb strength with the re- remains to be studied.The Bactrian camel lives in an gional demand for oxygen utilization is closely related. extreme natural environment for a long time,with Moreover,the distribution of Ngb was also found in a dry climate and strong ultraviolet rays.A long period the optic nerve of the Bactrian camel,providing clues of illumination leads to excessive production of ROS for further research on the physiological function of and synthesis of lipid peroxidation product MDA, Ngb in protecting the optic nerve under extreme living resulting in retinal damage.Such lesions are mainly conditions.The histological study on the expression located in the regions of mitochondria in the retina distribution of Ngb in the retina of the Bactrian camel (20),because the inner segment of the photoreceptor showed that the expression distribution of Ngb in the is rich in mitochondria,while the outer segment is retina of the Bactrian camel was similar to that ofother rich in polyvalent unsaturated fatty acids and prone to mammals,but also had its own characteristics.The the formation of MDA,which may cause the rod and positive expression of Ngb in the outer segment of the cone layer to be damaged by ROS and MDA.Fordel photoreceptor of the Bactrian camel's retina enriches et al.(12)studied sh-sy5y nerve cells and found that the morphological information of the distribution of

6 Med. Weter. environments, different specificity of antibody qual￾ity employed, and immunological detection. These changes lead to the different expression and localiza￾tion of neuroglobin which are suggested to influence the adaptive potential of an organism (1). The pigment cortex is located in the outermost layer of the retina, and its main function is to supply oxygen and nutri￾ents to the outer retina and absorb light scattering. Therefore, the pigment super cortex is vulnerable to the photochemical damage of visible light, which leads to the accumulation of reactive oxygen species (ROS), and then produces oxidative stress reaction, resulting in cell apoptosis. However, Ngb can remove excessive ROS, maintain the oxygen balance of tissue cells, and protect cells from light damage, which may also be one of the reasons for the different Ngb expression. Other results confirmed that oxygen affinity and enhanced auto-oxidation question the role in oxygen delivery and proposed that the neuroprotective effect might be due to radical scavenging or activation of protection mechanisms (4). Researchers are still debating the exact location of Ngb. Ran et al. (19) found that there were strong Ngb mRNA hybridization signals in the inner and outer nuclear layers of rats. However, the study of Xu et al. (25) showed that Ngb was not expressed in the outer nuclear layer. The results of this current study show that Ngb is not distributed in the outer nuclear layer, which is consistent with Xu’s report. Some studies documented that there might be certain differences in the spatial distribution of Ngb mRNA and protein in the retina, which makes the retinal structure of mammals to be partitioned. Ngb mRNA is mainly distributed in the cell body, while Ngb is distributed in the protru￾sion, suggesting that the Ngb protein has a transport process after translation. In the present research, the positive distribution of Ngb in the outer segment of the photoreceptor is rarely reported. The current results revealed the presence of Ngb in the outer segment of the photoreceptor. This result may be related to the different animal species or may be closely related to the function of the outer segment of the photorecep￾tor during the visual formation of the Bactrian camel under extreme environmental conditions. Whether Ngb is related to the adaptability of the environment remains to be studied. The Bactrian camel lives in an extreme natural environment for a long time, with a dry climate and strong ultraviolet rays. A long period of illumination leads to excessive production of ROS and synthesis of lipid peroxidation product MDA, resulting in retinal damage. Such lesions are mainly located in the regions of mitochondria in the retina (20), because the inner segment of the photoreceptor is rich in mitochondria, while the outer segment is rich in polyvalent unsaturated fatty acids and prone to the formation of MDA, which may cause the rod and cone layer to be damaged by ROS and MDA. Fordel et al. (12) studied sh-sy5y nerve cells and found that when Ngb protein expression increased or decreased, it was accompanied by a corresponding decrease or increase in H2 O2 content, indicating that Ngb was a ROS scavenger. On the other hand, Nayak et al. (16) found that Ngb produced by cultured nerve cells in vitro can reduce ROS levels, indicating that Ngb plays a certain role in ROS elimination. Similarly, the cur￾rent results have shown that Ngb positive expression of different intensity was found in the rods and cones and the super cortex pigment, indicating that Ngb may have an important repairing effect on retinal damage caused by light. However, further research is needed to confirm this report. The main function of the retina is visual conduction in which photoreceptors convert the received photochemical signals into electrical signals, and can be transmitted to the central nervous system through various neurons in the optic network membrane forming vision. The current study reported that Ngb was expressed in the cytoplasm, rods, cones, and other cells of the retinal microcytes and bipolar cells. However, other studies have shown (22) that there are a large number of synaptic connections of adjacent binary neurons between ganglion cells, bi￾polar cells and rod-cone cells to transmit information, which is one of the regions with the highest oxygen consumption in the retina and also the region with concentrated mitochondria, so it is very sensitive to oxidative damage caused by ROS. The high expression of Ngb as a ROS scavenger in mitochondria suggests that Ngb is maintaining a high oxygen consumption group that has more complex physiological functions in tissue metabolism. Ngb is expressed with different intensity in each layer of the retina, and its expression level is positively correlated with the demand for tis￾sue cells for oxygen. Moreover, Ngb is an endogenous neuroprotective agent, and the expression level sig￾nificantly increased due to hypoxia and ischemia of tissue cells (21). Roberts et al. (20) who employed the method of constructing a mathematical model showed that the effect of Ngb is not in the oxygen storage, but the oxygen may play a more important role in the models to predict photoreceptors in the retina anoxic area in the segment and inner plexiform layer which is involved in the functioning of high expression of Ngb area. The expression of Ngb strength with the re￾gional demand for oxygen utilization is closely related. Moreover, the distribution of Ngb was also found in the optic nerve of the Bactrian camel, providing clues for further research on the physiological function of Ngb in protecting the optic nerve under extreme living conditions. The histological study on the expression distribution of Ngb in the retina of the Bactrian camel showed that the expression distribution of Ngb in the retina of the Bactrian camel was similar to that of other mammals, but also had its own characteristics. The positive expression of Ngb in the outer segment of the photoreceptor of the Bactrian camel’s retina enriches the morphological information of the distribution of

Med.Weter. Ngb in the mammalian retina and provides valuable ported in the current study but differs from the results clues for the study of its function. of Xu et al.(24).The difference may be related to the Adult rabbits.Previous studies have shown that sensitivity of the antibodies and methods used Ngb was positively expressed in all layers ofthe rodent, Immuno-histochemical semi-quantitative analysis dog,and human retinas,excluding the pigmented cor- results showed that Ngb expression in rabbit retina tex and photoreceptors (5,23).The studies further layers has an obvious difference on the strength of the stated that Ngb is highly concentrated in the layers and section in the optic nerve fiber layer,cell layer and expression levels differ.Similarly,the current results inner plexiform layer,outer plexiform layer and opti- reported that Ngb expression was found in these re- cal sensors in segmental positive expression which has gions.The positive distribution of Ngb was observed the highest intensity.This is consistent with what in the outer reticular layer,outer membrane and the Schmidt et al.reports in the retina of mice(21).Studies inner segment of the photoreceptor,while no positive have shown that the optic nerve fiber layer,ganglion expression was observed in the outer segment of the cell layer,inner reticular layer,outer reticular layer and photoreceptor,retinal inner boundary membrane,optic inner photoreceptor segment of the mammalian retina nerve fiber layer,ganglion cell layer,inner reticular are the most concentrated parts of the retina in oxygen layer,and kernel layer.Up to the present,debate on consumption because these regions are the adjacent the exact localization and mechanism of Ngb remains layer of the synaptic connection between neurons and a focus among scientists.A study documented that Ngb mitochondria(27).These neurons are involved in the show sequence variation in vertebrate evolution,sug- passage of information through mutual synapses.The gesting conserved structures and functions,and an photoelectric signal conversion needs to consume large important role in the animal's metabolism(6).Li You amounts of oxygen,and Ngb in these regions is high- (15)et al.utilized immunohistochemistry to confirm ly expressed.These regions and the subcellular local- Ngb expression,and the results reported that the ex- ization of mitochondria are closely related since with pression of Ngb was observed in other layers except the expression of Ngb because the body carrying oxy- for the pigmentation cortex of rats and mice.However, gen can also be used by retinal neurons to satisfy the Ostoji et al.(17)applying similar methods found that demand of oxygen consumption.Additionally,Xu et Ngb was located in the retinae of humans.The expres- al.(24)found that there were more Ngb positive par- sion was mainly located in the upper epidermis of ticles distributed between the synaptic vesicles in the human and dog retinas.In the current study,Ngb presynaptic structure of the adult rat retina,which positive expression was observed in the superior cortex supported the hypothesis that Ngb was related to the of the retinal pigment in adult rabbits which is consis- oxygen consumption of the optic network membrane. tent with the observation of Ding et al.(10).There also This further suggested that Ngb could be secreted and exists a minimum difference in these results and the transported to the synaptic vesicles to complete com- difference may have originated from the sensitivity of plex physiological functions.The present study like- the antibody and the methodology employed. wise found that Ngb also had a high level of positive Additionally,to ensure the functionality of the retina, expression in the retinal pigment superior cortex of humans and most mammals through the retina and rabbits.Studies have shown that the inner folds of the choroid double loop to oxygen in a row,the inner plasma membrane of the pigment epithelial cells can retinal nutrition by the central retinal vascular system. expand the contact area between the pigment epithe- the outer retinal blood supply is mainly supported by lial cells and the choroidal capillaries,which is con- choroid cycle,and the circulation of choroid blood ducive to the selective transfer of nutrients and me- does not directly reach the photoreceptor cell layer, tabolites between the outer layer of the retina and the which must pass through a pigment in the cortex of choroidal capillaries (29).Moreover,epigallocytes oxygen to diffuse to the photoreceptors.This might be have the functions of vitamin A transport and metabo- one of the reasons for different Ngb expression. lism,drug detoxification,synthesis of melanin and Whether the presence of Ngb in the pigment super extracellular matrix,and phagocytosis and digestion cortex is produced by the pigment super cortex cells of membrane plates shed from the outer segments of or the transport and redistribution of Ngb produced in photoreceptors.These functions of pigment epithelial neurons remains to be verified by further experiments. cells all require higher energy requirements on them- As reported in the study of Ding et al.(11).Ngb is selves,suggesting that the high expression of Ngb in distributed in the mammalian retina outer nuclear the pigment epithelial layer may be related to the high layer which differs from the current research report. oxygen consumption needed to maintain normal func- The in situ hybridization histochemistry technique was tional activities.Studies have shown that Ngb plays used by Yan et al.(27)and Ostoji (17)to examine the a crucial role in the dynamic oxygen balance of the expression of Ngb in the inner and outer nuclear layer retina,and its existence can make the optic network in the retinae of human and rat.The results confirmed membrane endure longer under the condition of isch- that strong expression was found in the inner and emia and hypoxia(20).Shi et al.(22)found that Ngb outer nuclear layers which are similar to what is re- was rapidly increased after the lack of blood in the

Med. Weter. 7 Ngb in the mammalian retina and provides valuable clues for the study of its function. Adult rabbits. Previous studies have shown that Ngb was positively expressed in all layers of the rodent, dog, and human retinas, excluding the pigmented cor￾tex and photoreceptors (5, 23). The studies further stated that Ngb is highly concentrated in the layers and expression levels differ. Similarly, the current results reported that Ngb expression was found in these re￾gions. The positive distribution of Ngb was observed in the outer reticular layer, outer membrane and the inner segment of the photoreceptor, while no positive expression was observed in the outer segment of the photoreceptor, retinal inner boundary membrane, optic nerve fiber layer, ganglion cell layer, inner reticular layer, and kernel layer. Up to the present, debate on the exact localization and mechanism of Ngb remains a focus among scientists. A study documented that Ngb show sequence variation in vertebrate evolution, sug￾gesting conserved structures and functions, and an important role in the animal’s metabolism (6). Li You (15) et al. utilized immunohistochemistry to confirm Ngb expression, and the results reported that the ex￾pression of Ngb was observed in other layers except for the pigmentation cortex of rats and mice. However, Ostoji et al. (17) applying similar methods found that Ngb was located in the retinae of humans. The expres￾sion was mainly located in the upper epidermis of human and dog retinas. In the current study, Ngb positive expression was observed in the superior cortex of the retinal pigment in adult rabbits which is consis￾tent with the observation of Ding et al. (10). There also exists a minimum difference in these results and the difference may have originated from the sensitivity of the antibody and the methodology employed. Additionally, to ensure the functionality of the retina, humans and most mammals through the retina and choroid double loop to oxygen in a row, the inner retinal nutrition by the central retinal vascular system, the outer retinal blood supply is mainly supported by choroid cycle, and the circulation of choroid blood does not directly reach the photoreceptor cell layer, which must pass through a pigment in the cortex of oxygen to diffuse to the photoreceptors. This might be one of the reasons for different Ngb expression. Whether the presence of Ngb in the pigment super cortex is produced by the pigment super cortex cells or the transport and redistribution of Ngb produced in neurons remains to be verified by further experiments. As reported in the study of Ding et al. (11). Ngb is distributed in the mammalian retina outer nuclear layer which differs from the current research report. The in situ hybridization histochemistry technique was used by Yan et al. (27) and Ostoji (17) to examine the expression of Ngb in the inner and outer nuclear layer in the retinae of human and rat. The results confirmed that strong expression was found in the inner and outer nuclear layers which are similar to what is re￾ported in the current study but differs from the results of Xu et al. (24). The difference may be related to the sensitivity of the antibodies and methods used. Immuno-histochemical semi-quantitative analysis results showed that Ngb expression in rabbit retina layers has an obvious difference on the strength of the section in the optic nerve fiber layer, cell layer and inner plexiform layer, outer plexiform layer and opti￾cal sensors in segmental positive expression which has the highest intensity. This is consistent with what Schmidt et al. reports in the retina of mice (21). Studies have shown that the optic nerve fiber layer, ganglion cell layer, inner reticular layer, outer reticular layer and inner photoreceptor segment of the mammalian retina are the most concentrated parts of the retina in oxygen consumption because these regions are the adjacent layer of the synaptic connection between neurons and mitochondria (27). These neurons are involved in the passage of information through mutual synapses. The photoelectric signal conversion needs to consume large amounts of oxygen, and Ngb in these regions is high￾ly expressed. These regions and the subcellular local￾ization of mitochondria are closely related since with the expression of Ngb because the body carrying oxy￾gen can also be used by retinal neurons to satisfy the demand of oxygen consumption. Additionally, Xu et al. (24) found that there were more Ngb positive par￾ticles distributed between the synaptic vesicles in the presynaptic structure of the adult rat retina, which supported the hypothesis that Ngb was related to the oxygen consumption of the optic network membrane. This further suggested that Ngb could be secreted and transported to the synaptic vesicles to complete com￾plex physiological functions. The present study like￾wise found that Ngb also had a high level of positive expression in the retinal pigment superior cortex of rabbits. Studies have shown that the inner folds of the plasma membrane of the pigment epithelial cells can expand the contact area between the pigment epithe￾lial cells and the choroidal capillaries, which is con￾ducive to the selective transfer of nutrients and me￾tabolites between the outer layer of the retina and the choroidal capillaries (29). Moreover, epigallocytes have the functions of vitamin A transport and metabo￾lism, drug detoxification, synthesis of melanin and extracellular matrix, and phagocytosis and digestion of membrane plates shed from the outer segments of photoreceptors. These functions of pigment epithelial cells all require higher energy requirements on them￾selves, suggesting that the high expression of Ngb in the pigment epithelial layer may be related to the high oxygen consumption needed to maintain normal func￾tional activities. Studies have shown that Ngb plays a crucial role in the dynamic oxygen balance of the retina, and its existence can make the optic network membrane endure longer under the condition of isch￾emia and hypoxia (20). Shi et al. (22) found that Ngb was rapidly increased after the lack of blood in the

Med.Weter. optic network membrane,indicating that Ngb is very a similar method Ostojic I(17,18)showed that Ngb sensitive to hypoxia,suggesting that Ngb may play an is observed in all retina layers,especially the cortex important role in the adaptive protection of retinal of retina pigment in the human and dog.Despite these hypoxia,while Ding et al.(10)showed that in the acute reported references,there are still limited reports on the ischemic and hypoxic state the main concentration of retina of adult Tibetan sheep.The results of this study Ngb content in the retina of rats was the ganglion cell showed that there were positive distributions of Ngb in layer,and a small amount of Ngb content was also the inner boundary membrane,optic nerve fiber layer, found in the inner and outer netted layer.In addition, ganglion cell layer,inner reticular layer,inner layer, Yan et al.(27)explained that the expression of Ngb in outer reticular layer,outer membrane and an inner seg- the retinal tissues of rabbits was higher than that in the ment of the photoreceptor of the adult sheep retina but plain and subalpine environments under the hypoxic no positive expression was found in the outer segment environment,while Ngb in the plain area was only of the photoreceptor,pigment super cortex,and optic weakly expressed in the inner and outer plexus layers network membrane which is consistent with the find- and the inner segment of photoreceptors.The present ings of Bentmann et al.(2),but different from the ob- research results show that the expression of Ngb in the servation results of Ostojic et al.(17).The expression in optic nerve fiber layer and ganglion cell layer and inner these regions has shown to be strongly expressed with plexiform layer,outer plexiform layer,photoreceptor white mRNA of mixed-signal.Yan et al.and Ostojic inner segment and the intensity of expression of the et al.(18,27)also applied the immunohistochemistry highest in areas such as the pigment in the cortex, method to obtain the expression of Ngb and immune expressed in the kernel layer strength is weak,prompt response in the retina of rats and humans.The Ngb retinal optic nerve fiber layer and ganglion cell layer distribution in both rats and humans was consistent but and inner plexiform layer,outer plexiform layer and in their study Xu et al.(25)did not observe any Ngb optical sensors in segmental oxygen consumption,such distribution in the outer nuclear layer sub-cells of the as the concentration of the highest place on the degree adult rat retina.Similarly,the current reports also ob- of sensitivity of ischemia hypoxia,cortex cell me- served no positive Ngb expression in the outer nuclear tabolism active pigment,with special phagocytosis.At layer of adult sheep retina.To ensure the network of the same time,it also performs the function of bio- membrane function,human and most mammalian filtration of the sensory layer of the retina,which re- retina and choroid membrane double loop to continue quires a higher energy supply,so it is sensitive to the flow of oxygen.Central retinal nutrition blood ischemia and hypoxia the highest.Additionally,the pipe system and outer retinal blood supply are mainly inner core layer acts as a pathway for transmitting supported by the choroid cycle,and the circulation of photoreceptor cell impulses to ganglion cells,both of choroid blood does not directly reach the photoreceptor which have large oxygen consumption requirements. cells layer,which must pass through a pigment in the The sensitivity of ischemia and hypoxia was also cortex of oxygen to diffuse to the light receptors.The higher,while the areas with negative Ngb expression, results of the immuno-histochemical semi-quantitative such as the outer nuclear layer and the outer segment analysis showed that there were significant differences of the photoreceptor,were the least sensitive to hy- in the expression of Ngb in different layers of the sheep poxia.Therefore,the difference of Ngb expression in retina.The positive expression intensity was recorded each layer of the retina may also be correlated with as the highest in the dimension layer,the inner reticular the sensitivity of each layer to ischemia and hypoxia layer,the outer reticular layer,and the inner segment injury. of the photoreceptor.which was similar to the study Adult sheep.Existing research has shown that Ngb results of Schmidt et al.(21)who studied Ngb expres- is solely expressed in rodent,dog,and human retinae sion in the retinae of mice.Mammalian retinal nerve excluding the pigment in the cortex and light sensor fiber layer,inner plexiform layer,outer plexiform layer, section which contain no expression of Ngb.A study and optical sensors are the most important parts of conducted on the mouse retina found that Ngb is ex- oxygen consumption in retinal tissue(2,3),due to the pressed in all neurons especially the plexiform layers great amount of synaptic coupling between adjacent and the ellipsoid region of the photoreceptor inner seg- layer neurons and mitochondria.The neurons send ment(5),which is consistent with the current research information through mutual synapses and releasing the that showed expression in the plexiform layers.Ostojic photoelectric signal conversion.This process requires et al.studied the retina in canines (dog)and reported a large amount of oxygen consumption and the expres- that Ngb is solely expressed in the ganglion cell layer, sion of Ngb was showed to be higher in these regions inner and outer nuclear layers,inner and outer plexi- which are related to the subcellular localization of form layers,photoreceptor inner segments,and retinal mitochondria.The oxygen transported can be utilized pigment epithelium.By using immuno-histochemical by retinal neurons to satisfy the high oxygen demand Schmidt (21)and Li(15)confirmed that Ngb is not of the four layers.In the subcellular structure,the Ngb expressed in the layer of the skin color but can be found protein is not found in the mitochondria but around in other layers of the small rat retina,but by applying them,which has important implications for further

8 Med. Weter. optic network membrane, indicating that Ngb is very sensitive to hypoxia, suggesting that Ngb may play an important role in the adaptive protection of retinal hypoxia, while Ding et al. (10) showed that in the acute ischemic and hypoxic state the main concentration of Ngb content in the retina of rats was the ganglion cell layer, and a small amount of Ngb content was also found in the inner and outer netted layer. In addition, Yan et al. (27) explained that the expression of Ngb in the retinal tissues of rabbits was higher than that in the plain and subalpine environments under the hypoxic environment, while Ngb in the plain area was only weakly expressed in the inner and outer plexus layers and the inner segment of photoreceptors. The present research results show that the expression of Ngb in the optic nerve fiber layer and ganglion cell layer and inner plexiform layer, outer plexiform layer, photoreceptor inner segment and the intensity of expression of the highest in areas such as the pigment in the cortex, expressed in the kernel layer strength is weak, prompt retinal optic nerve fiber layer and ganglion cell layer and inner plexiform layer, outer plexiform layer and optical sensors in segmental oxygen consumption, such as the concentration of the highest place on the degree of sensitivity of ischemia hypoxia, cortex cell me￾tabolism active pigment, with special phagocytosis. At the same time, it also performs the function of bio￾filtration of the sensory layer of the retina, which re￾quires a higher energy supply, so it is sensitive to ischemia and hypoxia the highest. Additionally, the inner core layer acts as a pathway for transmitting photoreceptor cell impulses to ganglion cells, both of which have large oxygen consumption requirements. The sensitivity of ischemia and hypoxia was also higher, while the areas with negative Ngb expression, such as the outer nuclear layer and the outer segment of the photoreceptor, were the least sensitive to hy￾poxia. Therefore, the difference of Ngb expression in each layer of the retina may also be correlated with the sensitivity of each layer to ischemia and hypoxia injury. Adult sheep. Existing research has shown that Ngb is solely expressed in rodent, dog, and human retinae excluding the pigment in the cortex and light sensor section which contain no expression of Ngb. A study conducted on the mouse retina found that Ngb is ex￾pressed in all neurons especially the plexiform layers and the ellipsoid region of the photoreceptor inner seg￾ment (5), which is consistent with the current research that showed expression in the plexiform layers. Ostojic et al. studied the retina in canines (dog) and reported that Ngb is solely expressed in the ganglion cell layer, inner and outer nuclear layers, inner and outer plexi￾form layers, photoreceptor inner segments, and retinal pigment epithelium. By using immuno-histochemical Schmidt (21) and Li (15) confirmed that Ngb is not expressed in the layer of the skin color but can be found in other layers of the small rat retina, but by applying a similar method Ostojic I (17, 18) showed that Ngb is observed in all retina layers, especially the cortex of retina pigment in the human and dog. Despite these reported references, there are still limited reports on the retina of adult Tibetan sheep. The results of this study showed that there were positive distributions of Ngb in the inner boundary membrane, optic nerve fiber layer, ganglion cell layer, inner reticular layer, inner layer, outer reticular layer, outer membrane and an inner seg￾ment of the photoreceptor of the adult sheep retina but no positive expression was found in the outer segment of the photoreceptor, pigment super cortex, and optic network membrane which is consistent with the find￾ings of Bentmann et al. (2), but different from the ob￾servation results of Ostojic et al. (17). The expression in these regions has shown to be strongly expressed with white mRNA of mixed-signal. Yan et al. and Ostojic et al. (18, 27) also applied the immunohistochemistry method to obtain the expression of Ngb and immune response in the retina of rats and humans. The Ngb distribution in both rats and humans was consistent but in their study Xu et al. (25) did not observe any Ngb distribution in the outer nuclear layer sub-cells of the adult rat retina. Similarly, the current reports also ob￾served no positive Ngb expression in the outer nuclear layer of adult sheep retina. To ensure the network of membrane function, human and most mammalian retina and choroid membrane double loop to continue the flow of oxygen. Central retinal nutrition blood pipe system and outer retinal blood supply are mainly supported by the choroid cycle, and the circulation of choroid blood does not directly reach the photoreceptor cells layer, which must pass through a pigment in the cortex of oxygen to diffuse to the light receptors. The results of the immuno-histochemical semi-quantitative analysis showed that there were significant differences in the expression of Ngb in different layers of the sheep retina. The positive expression intensity was recorded as the highest in the dimension layer, the inner reticular layer, the outer reticular layer, and the inner segment of the photoreceptor, which was similar to the study results of Schmidt et al. (21) who studied Ngb expres￾sion in the retinae of mice. Mammalian retinal nerve fiber layer, inner plexiform layer, outer plexiform layer, and optical sensors are the most important parts of oxygen consumption in retinal tissue (2, 3), due to the great amount of synaptic coupling between adjacent layer neurons and mitochondria. The neurons send information through mutual synapses and releasing the photoelectric signal conversion. This process requires a large amount of oxygen consumption and the expres￾sion of Ngb was showed to be higher in these regions which are related to the subcellular localization of mitochondria. The oxygen transported can be utilized by retinal neurons to satisfy the high oxygen demand of the four layers. In the subcellular structure, the Ngb protein is not found in the mitochondria but around them, which has important implications for further

Med.Weter. 9 exploration of the physiological mechanism.It also 4.Brunori M..Vallone B.:Neuroglobin,seven years later.Cell Mol.Life Sci. enhances cell viability under hypoxia and under vari- 2007,64,1259-1268,doi:10.1007/s00018-007-7090-2 5.Burmester T.Weich B.,Reinhardt S..Hankeln T.:A vertebrate globin expressed ous types of oxidative stress in transgenic systems,but in the brain.Nature 2000,407,6803,520-523 does not appear to be strongly upregulated in response 6.Burmester T..Haberkamp M..Mit S..Roesner A..Schmidt M.Ebner B.. to stress(7).Additionally,Xu et al.(24)reported that Gerlach F.Fuchs C..Hankeln T.:Neuroglobin and Cytoglobin:Genes, Proteins and Evolution.IUBMB Life.2004,56,703-707,doi:10.1080/ the Ngb distribution in the presynaptic structure of 15216540500037257. rat retina between synaptic vesicles have more levels 7.Burmester T..Hankeln T:What Is the Function of Neuroglobin?J.Exp.Biol. of Ngb expression in males than females and further 2009,212,1423-1428,doi:10.1242/jeb.000729. supports the Ngb protein which is assumed to be as- 8.Chan S.,Saraswathy S..Rehak M.,Rao N.:Neuroglobin protection in retinal ischemia.Invest.Ophthalmol.Vis.Sci.2012,532,704-711. sociated with the retinal oxygen consumption.This 9.Costa B..Fawcett R,Li G.Y.,Safa N.N.:Orally administered epigallocatechin has suggested that Ngb protein can be secreted by the gallate attenuates slight induced photoreceptor damage.Brain research bulletin neurons and transported between synaptic vesicles to 2008.764,412-423 10.Ding G..Yan Z,He T.:The appearance of white and red eggs in rat retina dur- complete complex physiological functions.The expres- ing the death of acute ischemic ganglion cells.Zhonghua eye science journal sion of Ngb in different retinal layers may correspond 2010,467,590-596. to the sensitivity ofeach layer to ischemic and hypoxic 11.Ding G.:Research status of brain-globin in the retina.Journal of clinical injury.Ding et al.(11)found that in the acute ischemic ophthalmology 2010,186,573-575. 12.Fordel E.,Thijs L,Martinet W..Schrijvers D..Moens L.Dewilde S.:Anoxiaor and hypoxic state,the increase of Ngb in the cerebral of oxygen and glucose deprivation in sh-sy5y cells:astep closer to the unraveling the rat's retina was mainly concentrated in the nodule of neuroglobin and cytoglobin functions.Gene 2007,3982,114-122. 13.Geuens E.,Brouns 1..Flamez D.,Dewilde S.,Timmermans J..Moens L.: cell layer,and less expression observed in the inner A globin in the nucleus.J.Biol.Chem.2003,278,30417-30420 and outer reticular layer.The experimental results of 14.Greenberg D.,Jin K.,Khan A.:Neuroglobin:an endogenous neuroprotectant. the current study also reported that Ngb showed high Current opinion in pharmacology 2008,81,20-24. intensity in the optic nerve fiber layer,network layer, 15.Li Y..Liu H..Tong Y.:Immunohistochemistry of the distribution of brain globin in rat eyeball.Ophthalmic Research 2000,245,46 1-4 4. outer plexiform layer,and the light receptors.In areas 16.Nayak G.,Prentice M..Milton S.:Role of neuroglobin in regulating reactive such as the ganglion cells and kernel layer,the strength oxygen species in the brain of the anoxia-tolerant turtle trachemys scripta. of Ngb expression is weak.Other layers including Journal of neurochemistry 2010,1102,603-612. 17.Ostojic J..Gro-danic D.S..Syed N.A.,Hargrove M.S.,Trent J.T..Kuehn the optic nerve fiber layer,inner and outer plexiform M.H.,Kwon Y.H.,Kardon R.H.,Sakaguchi S.D.:Patterns of distribution layer,and optical sensors consumed the highest level of oxygen-binding globins,neuroglobin and cytoglobin in the human retina. of oxygen and they are the most concentrated areas of Arch.Ophthalmol.2008,12611,1530-1536. 18.Ostoji J.,Sakaguchi D.,De Lathouder Y..Hargrove M.S..Trent J.T.,Kwon high ischemia hypoxia sensitivity.In addition,the thin Y.H..Kardon R.H..Kuehn M.H.,Betts D.M..Grozdanic S.:Neuroglobin cell layer channels information to the brain while the and cytoglobin:oxygen-binding proteins in retinal neurons.Investigative retina kernel layer as impulse transfer photoreceptor ophthalmology visual science.2006,473,1016-1023 cells to access the ganglion cells.Both have a high 19.Ran J..Sun .Bi Expression of Neuroglobin mRNA in rat eyeball. Chongqing med..2003,47,1035-1036. demand for oxygen,and are highly sensitive to isch- 20.Roberts A.,Gaffney E..Luthert P.J.,Foss A.,Bryne H.:Retinal oxygen dis- emia hypoxia.The outer nuclear layer,photoreceptor tribution and the role of Neuroglobin.Joumal of mathematical biology 2016, outer segments,and pigment on the cortex of the brain 731,1-38. 21.Schmidt M.,Giessl A.,Laufs T.,Hankeln T,Wolfrum U.,Burmester T.:How showed Negative Ngb expression and express least does the eye breath?Evidence for neuroglobin-mediated oxygen supply in sensitive to the demand for oxygen. the mammalian retina.J.Biol.Chem.2783,1932-1935. 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Ngb physiological function in the retina and recorded 25.Xu O..Huang F,Wei Y.:Subcellular distribution of myoglobin expression in morphological data for further exploring the distribu- the retina of adult rats.International joural ofophthalmology.2009,1,35-37 (in Chinese with English abstract) tion of Ngb in normal mammalian retinas.However, 26.Yu Y.,CringleJ.:Oxygen distribution and consumption within the retina in the mechanism of Ngb involvement in damage repair vascularized and avascular retinas and animal models of retinal disease.Prog. after retinal injury remains to be studied further in Retina Eye Res.2001,202,175-208. 27.Yan G.,Yue H.,Duan W.:The effect of oxygen on the surface of membrane- molecular and tissue biology. encephaloglobin in rabbit optic network.New progress in ophthalmology 2010,307,618-621. 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Med. Weter. 9 exploration of the physiological mechanism. It also enhances cell viability under hypoxia and under vari￾ous types of oxidative stress in transgenic systems, but does not appear to be strongly upregulated in response to stress (7). Additionally, Xu et al. (24) reported that the Ngb distribution in the presynaptic structure of rat retina between synaptic vesicles have more levels of Ngb expression in males than females and further supports the Ngb protein which is assumed to be as￾sociated with the retinal oxygen consumption. This has suggested that Ngb protein can be secreted by the neurons and transported between synaptic vesicles to complete complex physiological functions. The expres￾sion of Ngb in different retinal layers may correspond to the sensitivity of each layer to ischemic and hypoxic injury. Ding et al. (11) found that in the acute ischemic and hypoxic state, the increase of Ngb in the cerebral of the rat’s retina was mainly concentrated in the nodule cell layer, and less expression observed in the inner and outer reticular layer. The experimental results of the current study also reported that Ngb showed high intensity in the optic nerve fiber layer, network layer, outer plexiform layer, and the light receptors. In areas such as the ganglion cells and kernel layer, the strength of Ngb expression is weak. Other layers including the optic nerve fiber layer, inner and outer plexiform layer, and optical sensors consumed the highest level of oxygen and they are the most concentrated areas of high ischemia hypoxia sensitivity. In addition, the thin cell layer channels information to the brain while the retina kernel layer as impulse transfer photoreceptor cells to access the ganglion cells. Both have a high demand for oxygen, and are highly sensitive to isch￾emia hypoxia. The outer nuclear layer, photoreceptor outer segments, and pigment on the cortex of the brain showed Negative Ngb expression and express least sensitive to the demand for oxygen. The reported results have suggested that Ngb may have an important physiological role in maintaining retinal oxygen homeostasis and participating in the repair mechanism of light damage in extreme survival to adapt to environmental conditions. The study further provides a theoretical basis for in-depth research of the Ngb physiological function in the retina and recorded morphological data for further exploring the distribu￾tion of Ngb in normal mammalian retinas. However, the mechanism of Ngb involvement in damage repair after retinal injury remains to be studied further in molecular and tissue biology. References 1. Alekseeva O. S., Grigorev J. P., Korzhewskij D. E.: Neuroglobin, an oxygen￾binding protein in the mammalian nervous system (localization and putative functions). J. Evolut. Biochem. Physiolog. 2017, 53, 249-258. 2. Anderson B.: Ocular effects of changes in oxygen and carbon dioxide tension. Trans Am. Ophthalmol. Soc. 1968, 66, 423-474. 3. Bentmann A., Schmidt M., Reuss S., Wolfrum U., Hankeln T., Brumester T.: Divergent distribution in vascular and avascular mammalian retinae links neuroglobin into cellular respiration. J. Biochem. 2005, 280, 21, 20660-20665. 4. Brunori M., Vallone B.: Neuroglobin, seven years later. Cell Mol. Life Sci. 2007, 64, 1259-1268, doi: 10.1007/s00018-007-7090-2. 5. Burmester T., Weich B., Reinhardt S., Hankeln T.: A vertebrate globin expressed in the brain. Nature 2000, 407, 6803, 520-523. 6. Burmester T., Haberkamp M., Mitz S., Roesner A., Schmidt M., Ebner B., Gerlach F., Fuchs C., Hankeln T.: Neuroglobin and Cytoglobin: Genes, Proteins and Evolution. IUBMB Life. 2004, 56, 703-707, doi: 10.1080/ 15216540500037257. 7. Burmester T., Hankeln T.: What Is the Function of Neuroglobin? J. Exp. Biol. 2009, 212, 1423-1428, doi: 10.1242/jeb.000729. 8. Chan S., Saraswathy S., Rehak M., Rao N.: Neuroglobin protection in retinal ischemia. Invest. Ophthalmol. Vis. Sci. 2012, 532, 704-711. 9. Costa B., Fawcett R., Li G. Y., Safa N. N.: Orally administered epigallocatechin gallate attenuates slight induced photoreceptor damage. Brain research bulletin 2008, 764, 412-423. 10. Ding G., Yan Z., He T.: The appearance of white and red eggs in rat retina dur￾ing the death of acute ischemic ganglion cells. Zhonghua eye science journal 2010, 467, 590-596. 11. Ding G.: Research status of brain-globin in the retina. Journal of clinical ophthalmology 2010, 186, 573-575. 12. Fordel E., Thijs L., Martinet W., Schrijvers D., Moens L., Dewilde S.: Anoxia or oxygen and glucose deprivation in sh-sy5y cells: a step closer to the unraveling of neuroglobin and cytoglobin functions. Gene 2007, 3982, 114-122. 13. 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