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.P<0.05 was considered significant.If there existed malin,glacial acetic acid,distilled water ratio:3:2:1:2) no significant difference in the MD,it was referred to as for 24 hours and stored at 4C.Later,the perfusion-fixed the negative control (P>0.05),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 was considered significant. If there existed no significant difference in the MD, it was referred to as the negative control (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