WITHDRAWN: Amelioration of retinal injury and improvement in associated memory by hUCB-derived cells is dose-dependent

doi:10.21203/rs.3.rs-4715086/v1

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WITHDRAWN: Amelioration of retinal injury and improvement in associated memory by hUCB-derived cells is dose-dependent

https://doi.org/10.21203/rs.3.rs-4715086/v1

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Background- The visual information transfers from retina to brain to amplify the neuronal signals resulting in visual perception. Damage in the retinal layer causes visual field defects such as cognition decline and memory loss. Although, various research investigations have attempted to reverse the damage using therapeutic interventions, however, these have not been successfully translated. In this study we aim to evaluate the efficacy of lineage negative stem cells derived from hUCB in reversing the retinal injury and visual memory by subretinal transplantation in mice model of laser injury.

Methods- Retinal injury was introduced in C57BL/6J male mice (24-28g) by using laser photocoagulation around the optic disc with defined parameters that disrupted the RPE layer of the retina. The 2 laser spots (2L) and 8 laser spots (8L) were created in each eye of the mice. The Fundus Fluorescein Angiography was used to confirm the establishment of varying degree of retinal injury. Around 50,000 stem cells were transplanted in each eye after 24 hours of laser injury. After 1 month, neurobehavioral assessments were carried out to estimate the visual-spatial memory using Morris Water Maze (MWM) and Passive Avoidance. Subsequently, the molecular assays including RT-PCR and immunohistochemistry were also undertaken.

Results- Fundus angiography confirmed presence of retinal vein leakage in the injury model in comparison to healthy control. To identify the cognitive deficits, the neurobehavioral assessments, based on Morris water maze and passive avoidance, were performed. The neurobehavioral tests show learning and cognitive improvement in the stem cell group in comparison to the injury group. Further, gene expression of neurotrophic factors, proliferative and apoptotic factors showed upregulated neuronal activity and possible neuroprotective role in rescue of retinal injury in stem cell group.

Conclusion-The Lin –ve stem cells rescued the injury and reversed the visual memory and retinal injury. The study shows that, with degree of injury, the number of lin –ve stem cells should be increased.

Visual memory

Lin-ve stem cell

Visual impairment

retina

retinal degeneration

Cognition

memory

Visual impairment and retinal neurodegeneration are inextricably linked and been associated to cognitive impairment and brain disorder. The retina serves as the primary relay in the visual pathway that transmits light impulses into axon potentials. Retinal dysfunction has been linked to degeneration of numerous visual pathway components, including the main visual region (V1), optic radiation, and lateral geniculate nucleus (1, 2). In visual pathway, stimuli from surroundings are processed by an extensive network of interconnected neurons that extends from the optic nerve in the eye to the visual processing area in the visual cortex in forebrain. All information is sent by nerve impulses, activated by photosensitive chemical processes in the retina. Indeed, retinal neurodegeneration has been linked to global and localized brain atrophy and cognitive impairment (3). Reduced visual stimulation may potentially cause retinal degeneration by concomitant reduction in neurotrophic factors (4). Studies have shown that visual impairment has been linked to an increased risk of cognitive impairment and retinal neurodegeneration, ultimately leading to cognitive decline and vision loss that results in disruption of the connection between visual and other brain regions (5–8). Various clinical trials and studies have been designed for the treatment of retinal degeneration but only early and recurring treatment using anti-VEGF therapies have been shown to slow down its progression and to provide systematic relief with frequent relapse episodes. Furthermore, stem cell therapies have been attempted to rejuvenate and repair the injured retina because the retina lacks innate regeneration capabilities (9–10). Numerous preclinical and clinical investigations have shown that the transplantation of stem cells results in substantial improvements. Stem cell therapies are currently being explored in phase 1 and 2 clinical trials for retinal degenerative disorders. Stem cells have been used as alternative to treat various retinal degeneration pathologies (11). To the best of our knowledge no permanent treatment has been shown to overcome retinal degeneration and visual impairment.

The present study investigated that whether lineage negative (lin -ve) stem cells derived from human umbilical cord blood were able to reverse visuospatial memory in laser induced retinal degeneration. We have previously reported that lin –ve stem cells can restore memory and enhance neurotrophic factors when administered in Amyloid beta injured mice. Here we hypothesize that lin –ve stem cells may mitigate retinal injury and improve visual memory.

All the experimental methods were approved by Institutional Animal ethical committee, Institutional stem cell research and Institutional Ethic committee. Institutional Animal Ethical Committee (105/IAEC/720), Institutional Ethics Committee (PGI/1EC/2021/000386) and Institutional Committee on Stem Cell Research and Therapy (IC-SCRT) of PGIMER, Chandigarh with approval no. PGI-IC-SCR-114-2020/3033.

Animals

C57BL/6J mice aged 24–26 weeks were included in the study of. The animals were housed according to standard 12 hours light/ 12 hours dark cycle maintained by central animal house facility,PGIMER. Special care was given to minimize the pain and discomfort to mice during the procedure. They were fed a standard diet and reverse osmosis (RO) drinking water was provided ad libitum. Body temperature was maintained with a heating pad to prevent cataract during the procedure. Only 2 animals were kept per cage. There were 6 groups in the study, in each group 10 animals were included (n = 60). In general, as per CPCSEA and Arrive guidelines minimum number of animals required for animal experimentation is 6, however, because of the high Mortality Rate in retinal degeneration mice model. The study followed ARRIVE 2.0 guidelines.

Establishment of laser injury model using laser photocoagulation

The study included eight groups subjected to a different degrees of laser injury (Fig. 1) around the optic disc and transplantation of lin –ve stem cells at a defined timepoints. Briefly, once the mice were anesthetized with ketamine-xylazine cocktail IP, the pupils were dilated with 1% tropicamide (Tropac) and phenylephrine. The slit lamp microscope (Appasamy Associate, India) was connected to a laser photocoagulator containing a diode-pumped with 532 nm argon green laser wavelength, double solid-state frequency (IRIDEX, USA), Once the site was visualized, laser shots were given around the optic disc in a circular fashion in each eye using a coverslip as a contact lens at standardized parameters at power of 200 milli-Watt (mW) during 100 milliseconds (ms), and size of laser spots was 100 µm. Each mouse received 2 and 8 laser spots per eye in the respective group

Fundus fluorescein angiography (FFA)

Animals were subjected to FFA after 24 hr of laser injury. Once the mice were anesthetized, the pupils were dilated using 1% tropicamide. The fluorescein dye 0.1% was injected in the caudal vein. Fundus imaging was done, and the leakage in the retinal blood vessels validated the laser mouse model injury.

Collection of human umbilical cord blood (hUCB)

The hUCB samples were collected from umbilical cord vein as per the inclusion and exclusion criteria. Under sterile conditions, the vein in the cord was identified, wiped with 70% ethanol and approximately 10 ml blood was collected in the EDTA (Ethylene diamine tetraacetic acid) coated vials and was gently mixed to prevent coagulation. The sample was transported on ice from the collection site to the laboratory for further processing.

Inclusion criteria:

The hUCB was collected from the umbilical cord of pregnant women (aged between 20–35 years) who underwent elective caesarean deliveries at the gestation period of ≥28 weeks. All the donors were screened, and an informed consent was obtained from the donors. The detailed procedures and objectives pertaining to the study were explained. The participants with Hepatitis B infection, Hepatitis C infection, HIV infection, Syphilis infection, untreated urinary tract infection (UTI), acute infection, unclean vaginal examination, fever, prolonged rupture of membrane (> 24h), foul smelling amniotic fluid, major congenital malformation in the new-born were all excluded from the study.

Isolation of Lin-ve stem cells derived from hUCB by Magnetic-activated cell sorting (MACS)

The mononucleated cells were isolated based on the principle of density gradient centrifugation. Briefly, the collected hUCB sample was layered over 2.5-3 ml Ficoll Histopaque solution (Sigma Aldrich, USA) in a 15 ml falcon tube in a ratio of 1:1. The sample was centrifuged at the speed of 1500 rpm for 30 mins. The total number of isolated mononuclear cells was counted using a haemocytometer. Further, the mononuclear cells (MNCs) were used to isolate the Lin-ve stem cells population using the MACS apparatus and a human lineage depletion kit (Miltenyi Biotec, Germany). The stem cells were separated into two populations: Lin-ve population and Lin + ve population.

Characterization of Lin-ve stem cells by flow cytometry

The Lin-ve stem cells derived from hUCB were characterized using flow cytometry to analyse the presence of stem cell markers in the isolated cells. The MNCs, Lin-ve, Lin + ve cell populations were tagged with cell surface markers specific to stem cell lineage. The cells were incubated with 20 µl FcR blocker for 30 minutes at 4ºC to block non-specific binding of antibodies to human Fc receptor-expressing cells such as B cells, monocytes, and macrophages. In the suspension, 20 µl CD34, 20 µl CD45 and 10 µl CD117 cell surface antibodies were incubated for 1h at 4ºC to detect specific cells within the heterogeneous population. After one hour, cells were resuspended in 1 ml of 1X PBS for washing using 2000 rpm for 2 minutes at 4ºC. The MNCs, Lin-ve and Lin + ve cells were acquired by flow cytometer (BD FACS-Canto II, Biosciences USA). The expression of CD34, CD45 and CD117 was analysed in each population using a specific gated system. The complete analysis was done using International Society of Hematotherapy and Graft Engineering (ISHAGE) guidelines. The guideline was designed to ensure accurate and reproducible enumeration of CD34⁺ stem cells using a Boolean gating strategy and specifically validated anti-CD45 and anti-CD34 conjugates. The results were acquired on the same day or the cells were fixed in 1% paraformaldehyde solution for next day data acquisition.

Absolute CD34 + cells count × 10⁶/L =

Absolute Leukocyte (LKC) (×10⁹/L) ×

(corrected average total no. of CD34 + events) × 1000

average total no. of CD45 + events

Transplantation of Lin-ve stem cells in mouse retina

The mice were anaesthetized by a ketamine-xylazine cocktail followed by dilation of eyes with tropicamide. Once the eyes were dilated, the mice were kept under a stereo zoom microscope (Leica, Germany). The transscleral approach which enables accesses to the subretinal space (SRS) through the choroid was used for sub retinal delivery of cells. Intraocular media is accessed through a sclerotomy in the limbus or pars plana using this route. A sclerotomy is created using a 22.5-degrees ophthalmic blade 0.5 mm away from the optic nerve. All the intraocular muscles were removed very finely. Next, a prick was made using a 25 G beveled needle, NanoFil™ RPE kit (World Precision Instrument, USA) was used with a 33 G blunt needle attached to a 10µl syringe containing stem cells inserted through the sclerotomy, with an angulation of 5–10 degrees, to reach the SRS. Isolated 50,000 Lin-ve stem cells from hUCB were suspended in 1X PBS, and cells were injected into subretinal space through the transscleral route into the mouse eye as per the groups described above. A detached bleb signifies successful transplantation.

Assessment of memory loss using neurobehavioral experiments

Rota rod apparatus

Rota rod was carried out to screen for any muscle-motor coordination deficits before subjecting them to neurobehavioral tasks. The animals that could not perform experiments were excluded from the study. The mice were allowed to rotate on the circular rod for 3 mins at 15 rpm. Three trials were done each day for 7 days before the Morris water maze experiment was performed. The latency to fall was recorded for each mouse.

Morris water maze (MWM) behavioral test

MWM was used to examine the effect of retinal injury on spatial memory and learning ability of mice. It is most frequently used for evaluation of spatial learning and memory in rodents such as mice and rats. The MWM tests impairments in visual short-term memory and visual–spatial abilities in small rodents. MWM is used to measure visual cues based on neurocognitive behavior consists of a circular water pool divided into four quadrants, with one hidden target placed in one of the quadrants. The protocol consists of six acquisition days and one retrieval day as 7th day. The water was filled into the tank up to the mark, and the quadrants were equally divided, named Q1, Q2, Q3, and Q4. The temperature and humidity were maintained between 22–25ºC and 40–60% throughout the experiment respectively. The system was connected with ANY-maze software and a video camera recorder. The visual cues were placed on the walls and pool side. The escape latency time was estimated for six acquisition days and the time spent by mice in each quadrant was measured on retrieval day (day 7th ). The escape latency time was compared between injury and stem cell transplantation groups. ANY-maze software includes various parameters such as escape latency time, mean speed and mean distance from platform to different quadrants, time spent by mice in each quadrant, and distance travelled from quadrant to platform.

Passive avoidance task to evaluate learning and memory

Passive avoidance is a fear conditioning test, which is associated with aversive learning and memory. The apparatus consists of two chambers differentiated as light chamber and dark chamber, separated by a gate known as guillotine door (). In the dark chamber, a 20-mA (milli-Ampere) current is applied. The mice neurobehavior goes against their intrinsic propensity to hide in dark places. The experiments continued for 3 days after Morris water maze experiments. On the first day (habituation day), the mice were allowed to move freely in both chambers. When a mouse entered the dark chamber with all four paws, the latency time was recorded, and the mouse was transferred back to the cage. On the second day (training day), mice were kept in the light chamber. After 30 seconds, the guillotine door was opened, and the time taken by each mouse to enter the dark chamber was recorded. Once a mouse entered the dark chamber, a mild foot shock of 20 mA was given, and the mouse was removed immediately from the chamber and shifted into the cage. On the third day (test day), the mice were kept in the light chamber and the time spent by mice in the light chamber to avoid foot shock in the dark chamber was recorded. The more time spent in the light chamber by mice implicates the learning behavior.

Animal Euthanization and enucleation of eyes

After neurobehavioral tests, the mice were euthanised using an overdose of Ketamine and xylazine i.p anaesthesia cocktail. The eyeballs were enucleated with the optic nerve and immediately stored at -80°C for further analyses such as cryosectioning, haematoxylin and eosin staining, immunohistochemistry and real-time PCR.

Cryosectioning of mouse eyes

The eyes were enucleated and embedded in a tissue freezing medium on a mould. The mounted block was placed in the cryotome, and sections with thickness of 8–12µm were taken on the poly L-lysine coated slides. The sections were kept at the RT for at least 1h before storing the slides at -20ºC so that the sections firmly adhered to the slide for further experiments such as immunohistochemistry and histology analyses.

Histological studies

Haematoxylin-Eosin (H & E) staining

Haematoxylin-Eosin staining is used to study histopathology and recognize different types of tissues or cells and their morphological changes. The staining was done to observe the distortion in retinal layer and loss of nuclei induced by laser injury followed by cresyl violet staining. Retinal sections were stained with cresyl violet acetate solution (Sigma Aldrich, Germany) for 15 mins. The imaging was done on EVOS Cell Imaging System M1000 (Thermofisher Scientific) and slides were stored at room temperature.

Immunohistochemistry (IHC)

The expression of retinal cell marker (Thy-1), neurotrophic factors (BDNF, CNTF), astrocyte marker GFAP and apoptosis marker Caspase-3 in the retinal layer were screened by immunofluorescence. The eye sections were air-dried at room temperature for

30 minutes. The sections were labelled using a PAP pen to demarcate the tissue for staining. Tissue sections were fixed for 20 mins with Histochoice (Sigma Aldrich, Germany). Sections on slides were washed thrice with PBS (pH 7.4) for 10 min. Epitope retrieval was done by incubating the sections with pre-warmed 2N HCl at 37°C for 30 mins. Sections on slides were washed thrice with 0.1 M borate solution for 10 mins. Slides were incubated with blocking serum (5% Bovine Serum Albumin in 1X PBS) in a humidified chamber for 1h at room temperature to block non-specific binding. The sections were labelled with conjugated primary antibodies diluted in permeabilization buffer (Biolegend) and incubated overnight at 4°C in a humidified chamber. Next day, the slides were washed with PBST (PBS with 0.1% triton X-100) thrice for 10 minutes each at room temperature and counter-stained with secondary antibody for 3h at 4°C in a humidified chamber. After incubation, the antibody was discarded, and the slides were gently washed thrice with 1X PBS. The slides were stained with 4,6-diamidino-2-phenylindole (DAPI) in 1:100 ratio for 5 min. Finally, the slides were dried and mounted with Fluorsave reagent to prevent any dye quenching. The sections were visualized under a confocal microscope (Olympus IX81, USA).

Retina isolation

Retina was isolated from the enucleated eyeballs under stereo zoom microscope (Leica). The eye was kept in a humidified sterile microplate using saline. The extraocular tissues were removed, and a small incision was made with fine forceps on the corneal- scleral junction. The lens was removed from the eye. The retina was scraped out carefully avoiding any contamination with vitreous humor and preserved in Trizin (GCC Biotech, India) until used for RNA isolation.

RNA isolation

RNA was extracted using the trizol method (GCC Biotech. India). The isolated retina was homogenized gently and mixed well in 1 ml trizol solution. Chloroform (200 µl) was added to 1 ml of trizol, and the solution was mixed vigorously by flicking up and down for 2–3 mins at room temperature. The sample was centrifuged at 12000g for 10 mins at 4ºC in an ultracentrifuge (Thermofisher). After centrifugation, two layers were formed containing aqueous layer and supernatant. The aqueous layer was transferred into a fresh microcentrifuge tube. Five hundred microlitre chilled isopropanol was added and incubated at room temperature for 5 mins, followed by centrifugation at 12000g for 10 minutes at 4ºC. The supernatant was discarded, and the pellet was carefully resuspended in 1ml 75% ethanol. The resuspended pellet was centrifuged at 7500g for 5 minutes at 4ºC and the supernatant was discarded. The pellet was allowed to air dry for 1 min and then the column was eluted with 30µl RNAse free water (Sisco Research Laboratories, India) and immediately kept at 4ºC further RNA concentration was measured. Nanodrop instrument (Eppendorf biospectrometer, USA) was used to estimate the concentration of RNA. Absorbance was taken at 260 nm wavelength. The RNA sample was immediately processed for cDNA synthesis.

cDNA synthesis

cDNA was prepared from the RNA samples immediately after the RNA isolation using the Verso cDNA synthesis kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The protocol was followed as mentioned in the commercially available kit. The cycle was set at 42°C for 30 minutes, 95°C for 2 minutes and infinity at 4°C, which was run on a Polymerase Chain Reaction (PCR) machine (Bio-Rad Laboratories, Inc.). After synthesis, the cDNA was immediately stored at -20°C.

Real-time PCR

Real-time PCR was used to quantitate expression of various genes including BDNF, CNTF, BCL-2, VEGF, Ki67 (Table 1). Specific primers were used to amplify the cDNA, and the PCR products were visualized. The amount of cDNA used was

100 ng for all the cDNA samples per reaction. Power up SYBR Green Master Mix reagent (Applied Biosystems, Foster City, California, USA) was used for evaluating the molecular expression of the markers. The default program of the ABi Step One software for ΔΔCt method for SYBR green reagents was used. The expression of the target genes was normalised with the β-actin housekeeping gene. The ΔΔCt method was used to calculate fold change.

Table 1

List of primers both forward and reverse for Real-time PCR.
S. No.	Target Gene	Make	Primer	Sequence
1.	Beta actin	Eurofins	Forward Reverse	5’-AGCCATGTACGTAGCCATCC-3’ 5’-CTCTCAGCTGTGGTGGTGAA-3’
2.	BCL-2	Eurofins	Forward Reverse	5’-GCCCTTCGGAGTTTAATCAG-3’ 5’-TACACTTGCACACACACGCT-3’
3.	VEGF	Eurofins	Forward Reverse	5’-CGATTGAGACCCTGGTGGA-3’ 5’-GTCTTTCTTTGGTCTGCATTCAC-3’
4.	BDNF	Sigma	Forward Reverse	5’-GCCCTTCGGAGTTTAATCAG-3’ 5’-TACACTTGCACACACACGCT-3’
5.	CNTF	Sigma	Forward Reverse	5’-GCGAGCGAGTCGAGTGGTTGTCTG-3’ 5’-TTAGCTTTCGGCCACCAGAGTGGAGAATTC-3’
6.	Ki67	Eurofins	Forward Reverse	5’-CAGTACTCGGAATGCAGCAA-3’ 5’-CAGTCTTCAGGGGCTCTGTC-3’

Quality assurance

All the experimental work was carried out according to the existing quality assurance system maintained in the Neuroscience Research Lab, Department of Neurology, PGIMER, Chandigarh. All data was cross validated with raw data, and cross linked to digitized data. Master coding was done for all samples. All the records such as Standard Operating Protocols (SOPs), Data Recording Sheet (DRS) and sample logs were prepared. The entire records pertaining to sample location, coding, data, and electronic files were assembled in the archive.

Statistical Analysis

Statistical analysis was done by calculating mean ± SEM. Each group included six mice. Normality was tested for each group. Comparison between the groups was done using ANOVA or Kruskal Wallis test in the study. The normally distributed data was analysed by using one-way ANOVA and repetitive ANOVA followed by LSD and post hoc analysis. p ≤ 0.05 was considered as statistically significant.

Confirmation of retinal injury model by Fundus fluorescence angiography

Fundus fluorescence angiography was carried out to confirm the establishment of injury model. Healthy group shows normal fundus with clear and functional retinal veins that supplies blood to the retina (Fig. 4A&B) whereas in the 2 laser retinal injury group, there was disruption of retinal vein that led to leakage in the retinal veins causing injury to the retina at two points (Fig. 4C&D). Similarly, in 8 laser injury group, the severe distortion in the retinal layer caused dysfunctioning of retina leading to partial blindness/ vision loss (Fig. 4E&F).

Characterization of retinal layer by histological staining

The retinal layers were identified using H and E & Cresyl violet staining. In healthy group, all the retinal layers were well organised and intact with each other (Fig. 5A&C) whereas in laser injury group, there was distortion in the retinal layers leading to loss of retinal ganglion cells and other cells in the retinal layers as shown in Fig. 5B&D.

Characterization of Lineage negative stem cells

The isolated cells were labelled with hematopoietic stem cell markers CD34 and CD45 using flow cytometry in order to characterize lineage negative stem cells (Fig. 6). A forward vs side scatter plot showed the distribution of CD45 positive cells in mononuclear cells (MNCs), Lin + ve and Lin-ve (Fig. 6A, B&C). Similarly, dot scatter plot depicted expression of CD34/CD117 in all three populations. About, 68.3% CD34 positive cells, an essential early hematopoietic lineage marker was obtained in the lin-ve cell population. The number of absolute CD34 cell counts in the Lin-ve population was found to be significantly high (p = 0.025) compared to Lin + ve cells, and the expression in mononuclear cells was found to be more as compared to Lin + ve cells (Fig. 7). This signifies that hUCB is a rich source of hematopoietic stem cells.

Neurobehavioral assessments show improvement in visual memory by lin –ve stem cells

MWM was carried out to measure the spatial memory. The average Escape Latency Time (ELT) was calculated on day 6 for 2 laser and 8 laser groups was 46.20 seconds and 19.57 seconds respectively as compared to healthy control mice (12.87 seconds) (Fig. 8A, B&C). The mice took more time to reach hidden platform in 2 injury groups, there was significant increase in ELT on acquisition trials from day 1 to 6 in comparison to the healthy control group (p = 0.024). To overcome the injury, stem cells were transplanted on the target site that may have potential to reverse the memory loss as seen in stem cell group, 2LSC that took very less time to reach hidden platform on day 5 and day 6 utlimately resulting in learning behaviour. The swimming pattern was recorded that showed impaired recovery in the injury group as compared to control group. In healthy control, the track plot showed that mice explored the maximum surface area using visual cues and took less time to reach hidden platform faster (Fig. 9A) whereas in the injury group, most of the mice spent time in the periphery and were unable to recall memory (Fig. 9 B, D). After stem cell transplantation, mice were able to retrieve memory and were able to reach hidden platform as shown in the Fig. 9 (C& E). In a retrieval trial on day 7, the hidden platform was removed from the target quadrant (Q1) and the time spent by mice in the target quadrants was calculated. We found that 8 laser injury groups spent significantly less time in the Q1 quadrant as compared to the healthy group (p = 0.015) (Fig. 10) while in 8 laser stem cell group, the mice spent significantly more time in Q1 quadrant as compared to injury group. This behavioural assessment has shown learning and memory restoration capacity in the transplanted groups. The search error index also revealed the maximum average distance of mice from the platform zone, indicating higher search error due to memory and visual impairment (Fig. 11 A, B & C) in injury group whereas stem cell group took less mean distance from platform. The swimming track recorded by the video tracking system showed thigmotactic behavior in the swimming pattern after 6 days of acquisition trials, this showed that the retinal injury affects brain function that leads to abnormal learning behavior. Further, stem cell transplantation reversed the injury and improve the memory/learning function. This data shows that retinal injury induces memory impairment in 2L can be rescue by stem cell therapy.

Passive avoidance

Passive avoidance is fear conditioning tasks is used to evaluate the learning and memory. Time spent by mice in the light chamber to avoid aversive stimuli was recorded in all groups. The operant behavior was recorded for each mouse on day 3. In injury groups, such as 2L and 8L injured mice have spent significantly less time in the light chamber and directly enters the darker region as compared to the healthy group (p = 0.004 and p = 0.004 respectively) thus showed the impairment in the learning and memory behavior. In the stem cell transplantation group, 2LSC and 8LSC mice had shown learning behavior as they spent significantly more time in the light chamber than dark chamber to avoid electric stock (p = 0.04 and p = 0.028 respectively (Fig. 12). The mice are nocturnal in nature. Therefore, the results show that in stem cell transplantation, group there was recall in the memory and evident from more time spent in light chamber.

Molecular effect of Lin-ve stem cells on mRNA expression

To evaluate the expression of Lin-ve stem cells derived from human UCB in laser-injured mouse models, neural, apoptotic and proliferative markers were studied. The retinal damage activates the apoptotic cascade, leading to neuronal cell death. We intended to assess the levels of neurotrophic, proliferative markers, apoptotic marker and angiogenic marker in the retina following hUCB Lin-ve stem cell implantation to determine the mechanism by which these cells exert therapeutic benefits in damaged mice eyes. Although all the genes have shown altered gene expression in injured mice, compared to the healthy control, the expression of BDNF, CNTF, Ki67 and BCL2 was found to be downregulated in comparison to healthy group. This expression shows that injury affects neurogenesis, neuronal survival, proliferative ability along with cell death as seen in the 2L and 8L group. In stem cell transplantation groups i.e. 2LSC and 8LSC, the expression of neurotrophic markers, proliferative marker and antiapoptotic marker was significantly upregulated that may promote neurogenesis, cell survival and proliferation of the cells leading to reversal of injury and functional pathway. The differences were observed in brain- derived neurotrophic factor (BDNF) (Fig. 13A), ciliary neurotrophic factor (CNTF) (Fig. 13 B), Ki67(Fig. 13 C) and BCL-2 (Fig. 13 D) expression. The expression of VEGF was not in any group (Fig. 13 E). The expression of BDNF was found to be significantly high as compared to 2L (p = 0.001). Similarly, in 8LSC the expression was significantly high (p = 0.003). The expression showed that stem cells have the potential to reverse the damage in the retina and restore vision. The fold change expression of CNTF was found to be significantly low in 2L and 8L as compared to the healthy control groups (p = 0.03). After stem cell transplantation, the expression was found to be significantly high in 2LSC as compared to 2L (p = 0.05). In case of Ki67, the fold change expression was downregulated in the 2L and 8L groups (p = 0.04 and p = 0.01) compared to healthy control. In stem cell transplantation groups such as 2LSC and 8LSC groups, the expression was more significantly upregulated (p = 0.001 and p = 0.002) compared to 2L and 8L groups. The expression of BCL-2 was found to be significantly upregulated in 2LSC (p = 0.013) and highly significant in 8LSC (p = 0.003).

Rescue of retinal injury after transplantation of Lin-ve stem cells, determined by immunohistochemistry

Upregulated expression of CNTF in post stem cell transplantation group may leads to neuroprotective role in rescue of injury.

The expression of BDNF in the 2L and 8L groups was not noticed, but after stem cell transplantation, we found its expression in the retinal layer as it is globally present in the retina (Fig. 14 A-E).

The quantitative analysis was carried out to measure BDNF expression in injured and stem cell transplanted groups. We report that its expression in the retina is downregulated in the injured mice such as 2L and 8L injury groups (p = 0.004, and p = 0.001 respectively) as compared to healthy control. Post stem cell transplantation in 2LSC and 8LSC, the expression was significantly increased (p = 0.014 and p = 0.001) (Fig. 14F). This shows that stem cell-enhanced BDNF expression was inclining towards neuronal survival.

Upregulated expression of CNTF in post stem cell transplantation groupmay leads to cell survival

After one-month of transplantation, retinal sections stained with CNTF specific antibodies were examined. The CNTF expression in 2L and 8L groups were low. After stem cell transplantation, we found significant upregulated CNTF expression in the retinal layer in 2LSCand 8LSC groups (p = 0.000, p = 0.000 respectively) (Fig. 15A-E). The quantitative analysis has shown downregulated expression of CNTF in injury mice in retina especially in all retinal layers and significantly upregulated in 2LSC and 8LSC transplanted groups (Fig. 15 F).

Downregulation of CASPASE-3 expression in post stem cell transplantation leads to cell survival

Caspase-3 expression was examined in all layers of the retina. The expression of caspase-3 in 8L group (p = 0.010) was found to be high that indicates the cell death in the injured sections but after stem cell transplantation in 8LSC group (p = 0.008) we found expression was diminished that showed Lin-ve stem cell potency to protect cells (Fig. 16A-F).

Downregulation of GFAP expression in post stem cell transplantation leads to cell survival

The expression of GFAP was s after retinal photocoagulation injury in the mice at different degrees of injury spots around the optic disc. After post transplantation of one-month, retinal sections were stained with a specific antibody to examine the expression of the protein GFAP (Fig. 17A-E).

The GFAP expression in 8L group (p=0.000) was found to have significantly high whereas after stem cell transplantation in 8LSC the expression was downregulated (p=0.000, p=0.013). This may involve recovery and reversal of retinal injury (Fig17 F).

Upregulation of Thy-1 expression in post stem cell transplantation leads to RGC survival.

Thy-1 is a retinal ganglion cell-specific marker that is required for the completion of the phototransduction process. The expression of Thy-1 in 2L and 8L group (p = 0.000) in the retinal ganglion cell layer was found to be significantly low, indicating the loss of RGCs and cell death in the injured sections as compared to the healthy control group (Fig. 18A-E). After stem cell transplantation in 2LSCand 8LSC group, we found presence of retinal ganglion cells in the GC layer as shown by fluorescence and in quantitative analysis (p = 0.014, p = 0.000 respectively) the expression of Thy-1 was significantly high in transplanted Lin-ve stem cells groups (Fig. 18 F).

Visual pathway plays important role that receive, integrate and process visual information relay from the retina. Visual signals transmit information from retina to visual cortex for executive functioning and decision making. Damage in the visual pathway causes visual field defects including decline in cognition, memory loss and neurodegeneration. Degeneration of ganglion cells leads to disruption in signalling pathways affecting the visual mechanism and resulting in visual impairment and retinal degeneration. This makes it essential to understand how changes in the retina affect the functioning and information process downstream in visual cortex pathways (12).

Individuals with retinal degeneration show decreased visual cortex activity and elevated associative areas outside the visual field, like coordination of frontal and supplementary eye fields like prefrontal cortex and intraparietal sulcus. Though neuroplasticity is preserved during adulthood for functions like learning and memory, the absence of these experience-dependent changes has also been considered through the visual system, particularly the visual cortex. Evidence suggests that preserved visual plasticity has an influence on perceptual learning, short-term visual deprivation, progressive blinding pathologies and visual restoration therapies (13).

The various interventions (retinal prostheses and pharmacological manipulations) are implemented monocularly to deepen understanding of the mechanisms regulating adult visual plasticity that is critical for visual restoration in late-blind individuals. Theoretically, the restored monocular signals might be gated at the cortical level if ocular dominance plasticity cannot be endorsed (13). Although many studies have been done and attempting to treat retinal degeneration, a permanent cure for retinal degeneration is lacking.

Several surgical procedures such as vitrectomy, retinal sutures, prone air fluid exchange, retinal incarceration has been employed for the treatment of retinal detachment and other retinal disorders but the recovery rate is poor and patients still suffer from various side effects (14–16). Experimental and clinical studies have provided evidence that vascular endothelial growth factor (VEGF) is a major factor in promoting neovascularization (17–19). Understanding the mechanism of visual and cognitive functions provide evidence to explore disease pathophysiology. Therefore, we have investigated the role of laser injury on visual spatial memory and its rescue by stem cell transplantation.

Laser induced as a model to study retinal degeneration

Animal models for induced choroidal neovascularization (CNV) have been the backbone for testing new therapies. Laser induced mouse models have been frequently used to understand retinal degeneration pathophysiology (20–21). This model provides a reproducible platform for preclinical bio-therapeutic screening aided by laser photocoagulator. The laser-induced endothelial cell activation in a retinal vein occlusion disease model has also been studied (22). Therefore, laser-induced retinal injury mouse model was established to damage targeted sites in the retina (23) to study the therapeutic efficacy of lineage negative stem cells (Fig. 3) that may restore the visual memory and molecular pathway that cause retinal degeneration and visual impairment. Further, injury was confirmed by fundus fluoresceine angiography (Fig. 4). Moreover, FFA is one of the most important diagnostic criteria for investigating the retinal disease condition (24–26).

Confirmation and efficacy of Lineage Negative stem cells that may have neuroprotective activity

The stem cells have proven the principle of therapeutics and its effects in repairing RPE. Previously, the study has shown that Lin-ve cells migrate, integrate and survive more than three weeks in laser injured micro-environment (27). Further, the role of Lin-ve Bone marrow-derived stem cells in the N-methyl-D-aspartate (NMDA) induced RGC depletion model and retinal ischemia model was also studied (28). Lin-ve stem cells derived from umbilical cord blood have the potential to reverse the memory loss and enhance neurotrophic factor to clear Amyloid β plaques (29). In retinal degeneration model, the cells from ciliary epithelium were delivered under the neural retina at the photoreceptor-RPE interphase subretinal route that showed protective role (30). However, in none of these studies visual-spatial memory or cortical plasticity was studied. However, study reveals that the hUCB-derived mesenchymal stem cells differentiated into RPE-like cells with RPE-specific retinal markers at both the mRNA and protein level. Moreover, these cells possess phagocytic activity and ability to secrete neurotrophic factors, which are the key characteristics of endogenous RPE-like cells (31). With the information obtained from previous studies, the investigation was done to assess effect of laser induced retinal injury on visual memory and its restoration by lin-ve stem cell transplantation. Therefore, stem cells were transplanted into subretinal space (the space between the RPE and the outer nuclear layer). This area has been extensively manipulated for cell and graft transplantation as the closest site to the photoreceptors and the RPE and helps escape immune rejection (32).

Stem cells ameliorate the retinal injury and improvement in visual functions

With developments in stem cell biology, significant progress has been achieved in creating retinal cells in recent years. Pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, can generate both sensory retinal neuron cells and retinal pigment epithelial cells in vitro (33). Retinal cell replacement using stem cell technology would be useful for restoring functioning retina, it has been used as a next-generation therapy for retinal degeneration (34–36). The number of studies have reported the efficacy of human stem cells in various animal models (37). The hUCB-derived cells were tested in rats, mice, and dogs, which were effective in brain disorders (37–38). The therapeutic effect of bone marrow mesenchymal stem cells on laser-induced retinal injury in mice, 1 × 10⁶ MSCs in 200 µL PBS was administered intravenously via tail vein injection. The study found that MSC therapy might have a neuroprotective role in restoring visual functions (39). The role of adult bone marrow-derived Lin-HSCs in the rescue from retinal degenerative mouse models by intravitreal transplantation into rd and rd10 mouse models. Lin-HSC has substantial vasculotrophic and neurotrophic effects, which lasts up to 6 months after therapy and is most effective when Lin-HSCs are injected before total retinal degeneration (40).

Further, studies have demonstrated that subretinal transplantation of MSCs in RCS rats slows retinal degeneration and retains retinal function (41). The studies have defined the protective role of stem cells derived from different sources at a specific interval (42–44).

Shirai et al., 2016 demonstrated the competence of hESC-retina in monkey models that may be helpful in treatment for long-term, functional studies of retinal transplantation (45). However, the study has shown that bone marrow derived mononuclear stem cells have therapeutic potential to treat retinal degeneration as seen in two animal models with different etiologies such as the RCS and the P23H-1 rats (46).

We aimed to transplant a Lin-ve stem cell derived from hUCB in the laser induced retinal injury and probe its effect on visuospatial memory and assess the retinal and visual memory outcome after transplantation.

Association of retinal injury with Cognitive impairment

Studies have demonstrated the relation of cognitive impairment with injury severity, as cognitive defects increase with the increase in injury severity in TBI mice model. The injury created by us was categorised into mild, moderate, and severe and was evaluated on various neurobehavioral tests. It was found that resulting cognitive impairment analysed by Morris water maze increased with injury severity (47). The studies have provided evidence that injury interferes with visuospatial memory and it impacts life activity (48–50). However, studies have shown that hUCB-derived stem cell therapy is a promising experimental treatment of disease (51). Previous studies have shown that stem cell treatment improves cognition (52–53). The neuroprotective effect of hUCB-MSC was studied by stimulating microglial neuroinflammation, therefore ameliorating disease pathophysiology and reversing the cognitive loss associated with Aβ deposition in AD animals (54). Furthermore, our behavioural tests have revealed that hUCB Lin-ve therapy improved learning and memory impairments, indicating the recovery of behavioural function and molecular expression.

Similarly, passive avoidance, was done to measure learning and spatial memory. In 2 laser and 8 lasers, there was significant memory loss compared to the healthy group, as mice spent much less time in dark regions to avoid electric shock. Post transplantation group, it was found that in 2 lasers + stem cell and 8 lasers + stem cell group transplantation group have shown significant improvement in learning and memory retrieval behaviour and found to reverse memory loss in the passive avoidance test (Fig. 4.13). This implies that milder injury can be rescued by Lin-ve transplanted stem cells, whereas severe damage leads to more distortion in the retinal layer, which ultimately leads to disruption in retinal function and causes visual memory loss.

Therefore, in the present study, we determined the effects of hUCB-derived Lin-ve stem cells on retinal degeneration and the recovery of visual function after transplantation in a rodent model. Furthermore, our behavioral experiments show that treatment with hUCB Lin-ve stem cells can improve the performance in memory tasks (Fig. 19).

Thus, stem cells have potential to rescue from the damage and lead to an increase in the expression of neurotrophic factors. Studies have shown that BDNF, CNTF, bFGF (basic fibroblast growth factor) have neuroprotective effects and pro cell survival activity in retinal ischemia-induced rat models, these molecules interact with each other (55). Rhee et al., (2022) has shown that by boosting aerobic glycolysis and enhancing anabolic activities, CNTF considerably influences the metabolic state of degenerating retina. These results shed light on the molecular mechanisms causing increased neuronal survival and new treatments for retinal degeneration (56).

Furthermore, BDNF signalling is needed for both embryonic and adult neurogenesis and our findings suggest that treating with hUCB Lin-ve increases endogenous BDNF level as well as the restoration of behavioural function. Thus, our research establishes causal relationships between behavioural tasks and hUCB Lin-ve induced secreted BDNF signalling, which may initiate intracellular signalling in the retina, elevating BDNF expression and contributing to cognitive function recovery in the injury model (Fig. 19).

However, studies have shown that conventional approaches with endogenous growth factors enhance the effectiveness of stem cell therapy. Glial cells such as astrocytes, oligodendrocytes, and microglia are secreted all these factors play an important role in neurodevelopment, growth and survival as the brain develops. Endogenous neurotrophic factors such as glial cell-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and nerve growth factor are expected to contribute to the support of the grafted nerve cells (57). Recent reports suggest that a novel small molecule BT13 binds RET to activate its downstream signaling cascades BT13 protects DA neurons from neurotoxin-induced cell death in vitro (58). Furthermore, BT13 activates DA signaling through RET and promotes DA release in the striatum in vivo, suggesting that BT13 is a candidate for the treatment of PD that may be suitable for cell removal excrete well and warrant further investigation (59). Advancement in stem cell biology and drug discovery has future insights. Although the studies have also shown that there is no need to repeat clinical trials in cell therapy without expansion. In fact, nothing new has been discovered about the safety or efficacy of stem cells after repeating clinical trials. Clinical trials should be conducted only on certain stem cell compounds, or in situations where the phenotype or genotype of stem cells may change (60)

Similarly, proliferative marker expression was analysed in the laser group and stem cell group (Fig. 4.18). It was found that retinal injury affects the proliferative ability and the expression of Ki67 was decreased in 2 laser injury i.e., mild injury and in 8 laser injury (severe injury), after stem cell transplantation in a respective group, it was found the expression was high as transplanted stem cells were able to proliferate in the surrounding environment (61).

Further, studies have demonstrated proliferative and differentiative properties of hUCB-derived stem cells (62). The VEGF protein level was compared with the injury group and stem cell. The effect of VEGF on retinal injury was found comparable.

Many studies have found that VEGF regulation is involved in disease progression, indicating that VEGFs secreted by cells initiate and promote pathological choroidal and retinal neovascularization processes in AMD (63).

At mRNA level, cell death markers were also analysed. The expression of Caspase 3 and GFAP was found significantly upregulated that indicates severe damage as compared to healthy control (Fig. 4.23 and 4.24). After stem cell transplantation, the expression was downregulated, which means the stem cell can rescue the damage and is better able to overcome retinal injury (Fig. 19). The present study has shown the involvement of astrocytes and Muller cells in the process. These results provide new insight into the gliosis-based mechanism in retinal degeneration regeneration.

Similarly, the RGC survival was analysed by Thy-1 expression, whose downregulation indicates loss of RGC in all degrees of injury as verified by immunohistochemistry (Fig. 4.25). This means that mild injury affects the retinal function and can be rescued by stem cell transplantation, as shown by recovery of lost RGC in the 2, 4 and 8 laser Lin-ve stem cell transplantation group. The therapeutic use of pluripotent cell-derived RGCs by grafting the cells in a healthy environment using suitable technological methods (64). The antiapoptotic marker BCL-2 was decreased in 2 lasers and 8 laser groups but the stem cell has shown improvement in the expression to indicate less cell death in 2 lasers + stem cell group and 8 laser stem cell group (Fig. 4.20). The studies have stem cells that can rescue the laser damage derived from bone marrow (65–66). Various studies have revealed that UCB-derived stem cells can proliferate into retinal cells that are further involved in repair mechanisms. The studies have shown that milder injury can be healed faster as compared to severe injury. Therefore, our research shows that stem cells could repair the damage and rescue the injury at a mild degree compared to severe injury (Fig. 19). The results suggest that stem cell therapy may induce a neuroprotective role in the rescue of retinal damage that restore visual function and visuospatial memory. UCB derived stem cells have good therapeutic effect in treating various clinical disorders (67–68)

The current study shows Lin-ve stem cells involvement in the rescue from visual impairment and its impact on visual-spatial memory. However, further investigation can be taken up to study the effect of retinal injury on the hippocampus in the brain and how stem cells replenish the memory functioning in the brain morphology and to analyse the brain section to investigate the morphological changes involved in the visual pathway. It will be interesting to dissect the putative molecular pathway involved in hippocampus changes by retinal injury in future studies for further insights.

Limitation of the study

To analyse functional and morphological changes using, electroretinograms (ERG) and optical coherence tomography (OCT) was not included.

Acknowledgement

We acknowledge Mr. Arun Kapil from Department of Ophthalmology, PGIMER for Fundus Fluorescein Angiography FFA imaging

We declared that no Artificial Intelligence used in this study.

Ethics approval and consent to participate

Name of the Institutional approval committee

All the ethical approvals were taken from Postgraduate Institute of Medical Education and Research (PGIMER) institute.

Ethical Approval-

Title of approved project:

The title of the study was approved by ethical committees from the Institutional Ethics committee (PGI/1EC/2021/000386) dated on 28.01.2021, Institutional Animal Ethical Committee (105/IAEC/720) dated on 12.12.2019, and the Institutional Committee on the Stem Cell Research and Therapy (PGI-IC-SCR-114-2020/3033) dated on 23-09-2020 from PGIMER, Chandigarh, India.

Consent to participate- All the donors were screened, and an informed consent was obtained from the donors. The detailed procedures and objectives pertaining to the study were explained.

Consent for publication

All authors confirm their consent for publication.

Source of Funding- None

Conflict of Interest- None to be declared.

Data Availability-The datasets for this article are not publicly available because of data protection regulations. Access to data can be provided on request in accordance to journal guidelines.

Author’s Contribution

PM- Performed the experiments, Analysed the data and wrote the manuscript

AA- Conceived and designed the experiments, Conceptualized the study, Review and edit the manuscript

MR- Edit and Analysed the data

JS- Review and Edit the manuscript

PKS- Review and Edit the manuscript

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WITHDRAWN: Amelioration of retinal injury and improvement in associated memory by hUCB-derived cells is dose-dependent

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Abstract

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Animals

Establishment of laser injury model using laser photocoagulation

Fundus fluorescein angiography (FFA)

Collection of human umbilical cord blood (hUCB)

Inclusion criteria:

Isolation of Lin-ve stem cells derived from hUCB by Magnetic-activated cell sorting (MACS)

Characterization of Lin-ve stem cells by flow cytometry

Transplantation of Lin-ve stem cells in mouse retina

Assessment of memory loss using neurobehavioral experiments

Rota rod apparatus

Morris water maze (MWM) behavioral test

Passive avoidance task to evaluate learning and memory

Animal Euthanization and enucleation of eyes

Cryosectioning of mouse eyes

Histological studies

Haematoxylin-Eosin (H & E) staining

Immunohistochemistry (IHC)

Retina isolation

RNA isolation

cDNA synthesis

Real-time PCR

Quality assurance

Statistical Analysis

Results

Confirmation of retinal injury model by Fundus fluorescence angiography

Characterization of retinal layer by histological staining

Characterization of Lineage negative stem cells

Neurobehavioral assessments show improvement in visual memory by lin –ve stem cells

Passive avoidance

Molecular effect of Lin-ve stem cells on mRNA expression

Rescue of retinal injury after transplantation of Lin-ve stem cells, determined by immunohistochemistry

Upregulated expression of CNTF in post stem cell transplantation groupmay leads to cell survival

Downregulation of CASPASE-3 expression in post stem cell transplantation leads to cell survival

Downregulation of GFAP expression in post stem cell transplantation leads to cell survival

Discussion

Laser induced as a model to study retinal degeneration

Confirmation and efficacy of Lineage Negative stem cells that may have neuroprotective activity

Stem cells ameliorate the retinal injury and improvement in visual functions

Association of retinal injury with Cognitive impairment

Conclusion

Limitation of the study

Declarations

References

Status:

Version 1