The optimal dose of human umbilical-cord mesenchymal stem cells (hUC-MSCs) on hypoxic ischemic brain damage(HIBD) rats via intranasal transplantation

doi:10.21203/rs.3.rs-4708272/v1

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The optimal dose of human umbilical-cord mesenchymal stem cells (hUC-MSCs) on hypoxic ischemic brain damage(HIBD) rats via intranasal transplantation

https://doi.org/10.21203/rs.3.rs-4708272/v1

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Background: Hypoxic ischemic brain damage(HIBD) cause permanent damage to the central nervous system (CNS), including mental retardation, aphasia, epilepsy, cerebral palsy, and developmental delays. Recently, human umbilical cord mesenchymal stem cells (hUC-MSCs) have emerged as a promising cell therapy in HIBD. The optimal dose of stem cell therapy remains open.

Methods: Previously, we conducted a study to identify that the best route of administration of hUC-MSCs to HIBD rat model is intranasal transplantation.

In this study, we conducted the comparison experiments for the optimal dose of MSC via the best route of administration, intranasal transplantation. Four different doses (0.5*10⁶, 1.0*10⁶, 1.5*10⁶, 2.0*10⁶) of hUC-MSCs were injected to HIBD rats (obtained HIBD at 7^th day after birth) on the10^th day after birth.

The body weight, hanging wire test and Morris Water Maze test were used on the 28^th day of different groups of rats to compare their motor function, memory, and learning ability. The HE staining was used to compare the morphological differences of brain tissues of rats in different groups. Moreover, the immunohistochemistry was adopted to compare the morphology and number of astrocytes and microglia in cortex and CA1, CA3 region of the brain of different groups.

Results: All the four different doses of hUC-MSCs treatment significantly improved their body weight, the motor function, memory, learning ability, the morphology and number of astrocytes and microglia in cortex and CA1, CA3 region of HIBD rats. The 1.5*10⁶ of hUC-MSCs group significantly outperform other three groups (0.5*10⁶, 1.0*10⁶, 2.0*10⁶) on both neurobehavioral motor function improvement and morphological improvement, i.e., reducing the number of astrocytes and microglia in cortex and CA1, CA3 region of HIBD rats. The study suggests that 1.5*10⁶ of hUC-MSCs was the optimal dose for rats who obtained HIBD at 7^th day after birth and injected by intranasal transplantation on the 10^th day after birth.

human umbilical cord mesenchymal stem cells (hUC-MSCs)

Hypoxic-ischemic brain damage (HIBD)

Intranasal transplantation

optimal dose of MSC

Hypoxic-ischemic brain damage (HIBD) is caused due to insufficient oxygen and blood supply in perinatal period, with annual incidence 1–8‰(Dumbuya et al. 2021). Every year, there are 0.75 million infants in the world suffers from moderate and severe HIBD, with irreversible brain damage and loss of neuronal function, dysfunction in memory, learning and movement (Shankaran et al. 2005; Douglas-Escobar and Weiss 2015; Zhou et al. 2021; Victor et al. 2022). However, the treatment about cognitive functional recovery remains debatable.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) have been used as a treatment of many diseases such as arthritis, diabetes and its complications, brain injury, heart diseases, cerebrovascular disease, respiratory diseases, viral infections, and other diseases because of following reasons. Firstly, hUC-MSCs are derived from the umbilical cord and have the potential to self-renew and pluripotent. They can proliferate and differentiate into different kinds of cell types under certain conditions. Secondly, they are easily harvested, isolated, cultured, amplified and purified. Thirdly, hUC-MSCs surface antigens are not prominent, rarely cause rejection, and the matching requirements with the body are not strict, which is easy to use in allotransplantation(Li et al. 2018; Guan et al. 2019; Xie et al. 2020). They function well on inhibiting the proliferation of pro-inflammatory cells and reducing inflammation (Guo et al. 2022; Li et al. 2022; Rodríguez-Eguren et al. 2022; Li et al. 2023); reducing oxidative stress and inhibiting cell apoptosis (Dong et al. 2021; Li et al. 2022); producing differentiated cells for tissue repair (Hoang et al. 2022; Yang et al. 2021; Guo et al. 2022; Rodríguez-Eguren et al. 2022; Zhou et al. 2022; Hou et al. 2023). In molecular level, they can secrete exosomes such as miRNA(Yuan et al. 2021; Shi et al. 2022), lncRNA(Lee et al. 2022; Yu et al. 2022) and circRNA(Dong et al. 2019; Ma et al. 2022), modulating target genes to restore neuron function (Xie et al. 2020).

HUC-MSCs is also a promising treatment to HIBD on neuronal functional recovery. It shows that 24 h after HIBD injury, the neuroinflammation cause circulating pro-inflammatory cytokines, and activate microglia(Serdar et al. 2020; Fleiss et al. 2021; Serdar et al. 2019). Besides, astrocytes also have a variety of effects, including neurotransmitter stability, synonym formation and removal, synovation regulation, neuropathy coupling, and blood brain barrier(Endo et al. 2022). However, they express and role differently in different areas of brain.

Although hUC-MSCs have been shown effectively on HIBD functional recovery, it remains open which are the best clinical practice, such as route of administration, dosage, and timing (van Velthoven et al. 2012). Previously, we investigated the optimal route of administration of hUC-MSCs on HIBD rats and showed that the functional improvement and pathological repair effect of intracerebral (IC) and intranasal (IN) administration were better than those of intravenous (IV) administration stem cells. For the clinical practice, the intranasal (IN) administration is non-invasive and more acceptable by patients than intracerebral (IC), so we transplant hUC-MSCs to HIBD rats by intranasal (IN) administration in this study. Currently, 72 hours after acute hypoxic-ischemic brain injury is recommended as the best time, since stem cells can promote the differentiation of neurons and oligodendrocytes, which lead to reduce multiplication of inflammatory cells, and secondary brain injury(van Velthoven et al. 2010).

The clinical trials of hUC-MSCs in children with cerebral palsy are in progress, while the dose of treatment is still controversial. It has been reported that different doses of stem cells effects differently on neuronal regeneration, reducing the area of cerebral infarction, and recovering the cognitive function of animals. Therefore, it is necessary to explore the optimal dose through animal models to provide better guidance for clinical treatment. Animal studies have shown the dose of stem cells administered to HIBD rats ranges from 1 * 10⁵ to 1*10⁷. We thus designed four doses (0.5*10⁶, 1.0*10⁶, 1.5*10⁶, 2.0*10⁶) of hUC-MSCs to inject HIBD rats via intranasal (IN) administration and 72 hours after HIBD injury. Neurobehavioral experiments were conducted to compare the cognitive and motor function recovery by hUC-MSCs. The HE staining was used to compare the morphological differences of brain tissues in different groups. The pathological repair effect of astrocyte and microglia were compared by the immunohistochemistry of glial fibrillary acidic protein (GFAP) and ionized calcium-binding linker molecule-1 (IBA1) on in cortex and CA1, CA3 region of the brain of HIBD rats. The results suggests that 1.5*10⁶ of hUC-MSCs was the optimal dose for rats who obtained HIBD at 7th day after birth and injected by intranasal transplantation on the 10th day after birth.

2.1 Animals and the HIBD Model

All the animal experiments are approved by the ethical review of animal welfare in Hubei University of Medicine. The rats were kept in a constant temperature and humidity room with 12 hours of alternating light and dark, with food and water. The HIBD rat model was established according to (Vannucci and Vannucci 1997): the SD rats at 7th day after birth were anesthetized with isoflurane, and their right common carotid artery were permanently ligated. After surgery, rats were situated in a humidified hypoxic tank filled with 8% oxygen + 92% nitrogen and a flow rate of 1.5 L/min for 2.5 h. The preliminary screening was performed to confirm successful modeling. After successful modeling, the HIBD rats were randomly assigned to HIBD group, and different doses of hUC-MSCs groups: 0.5* 10⁶ (MSC1), 1.0* 10⁶ (MSC2), 1.5* 10⁶ (MSC3), and 2* 10⁶ (MSC4) group. The four MSC groups were injected hUC-MSCs at 72 h after HIBD via intranasal injection. A sham group of rats were also prepared for comparison.

2.2 Human Umbilical Cord Mesenchymal Stem Cells (hUC-MSCs)

The human umbilical cord mesenchymal stem cells (hUC-MSCs) were obtained from the Stem Cell Center of Taihe Hospital, Shiyan City, and sponsored by Shenzhen Beike Biotechnology Co., LTD. The 2nd to 3rd generation of stem cells obtained from the company, were placed in an incubator with 5% CO2 for 3 minutes at 37℃, then inoculated into 10*10cm petri dish containing 10% FBS medium at a cell concentration of 1*10⁶/L and cultured in an incubator. After 8 hours, cell adherence was observed under a microscope to prepare for the first fluid change. When the cells continued to grow to 80% of the entire dish, passage started, and the cells were cleaned with fresh PBS for 3 times. 1.5mL 0.25% pancreatic enzyme were added in the incubator for 2 minutes. When all cells become round under a microscope, pour 5mL of complete medium to terminate digestion. A 1mL pipette gun was used to get the cells gently blown clockwise, and put them containing the complete medium into a 15mL EP tube centrifuged for 2 minutes. Discarding the supernatant, the cells were evenly spread in the petri dish. The culture medium was changed every 48 hours with the cell status checked. The 3rd to 5th passages cells were used to inject to HIBD rats.

2.3 Intranasal Injection of hUC-MSCs

0.2ml 10% chloral hydrate was injected intraperitoneally to the anesthesia rat at 72 hours after the HIBD modelling. Different MSC groups of supine rats, MSC1, MSC2, MSC3, and MSC4, were injected 2uL, 4uL, 6uL, 8uL of hUC-MSCs, correspondingly, into the nasal cavity slowly by a micro-syringe. The rats were kept supine for 5 minutes and returned to their mother when they recovered. As a comparison, the sham and HIBD group rats were injected of normal saline intranasally.

2.4 Hanging Wire Test

The hanging wire test was used to evaluate the neuromuscular development of different groups of rats. The test animal was first put on the top of a wire cage lid. We gently shake it a few times and then turn the lid over. The rat's forelimbs grip the lid tightly to avoid falling. A box filled with sawdust was put 20 cm away from the lid at the bottom to get the rats if they fall. The time of the test rat hanging on the lid until they fall into the box was measured using a stopwatch. Each rat was tested three times, and a one-minute break was performed between adjacent two tests. The average hanging time was then used to evaluate the rat’s ability of motor function.

2.5 Morris Water Maze

The Morris water maze (Morris 1984) was usually used to evaluate the learning and memory ability. A big black pool with diameter of 1.8m, depth of 0.6m was used in the experiment. The depth of water in the pool was kept 0.4m and a heating equipment was used under the pool to keep the temperature of water at 23 ± 2℃. Four quadrants were labelled on the wall of the pool. A planform with diameter of 10cm was put on the 3rd quadrant of the pool. The top of the pool was added blackout curtains to avoid light onto the water. A camera was put on the top of the pool to record the movements of the rats. The rat was put into the pool one day before the experiment to adapt to the environment. Place navigation tests were carried out in six days. Each rat was trained three times, with interval of more than 30 minutes, every day.

On the first day, the rat approached the water from a non-target quadrant, if it can reach the planform within 60s, we put the rat on the planform for 5s and the camera will record its movement trajectory, otherwise, if it cant’s reach the planform within 60s, we put it on the planform for 30s. The rat was sent back to its cage after drying off water.

On the 2nd and 5th day, the rat approached the water on the different non-target quadrant from the day before, and then tested in the same way with the 1st day.

On the 6th day, the planform was removed, the rat was stared from the 1st quadrant, and record its movement trajectory, the number of reach the target quadrant, and the time of staying the target quadrant within 60s.

2.6 Body Weight Measurement

At postnatal day of 7th, 21st and 28th, each rat's body weight was measured between 10am and 12am to avoid the effects of body weight change between days and nights.

2.7 Hematoxylin-Eosin Staining

The brains of six groups of rats were taken immediately 24 h after the behavioral experiments. The brains were then fixed in 10% formalin, then embedded with paraffin, and finally cut into 4 mm slices. The dorsal coronal surface of hippocampus (5µm thickness) was taken, and the brain sections of young rats (20µm) were soaked with hematoxylin solvent for 5 mins, then washed with pure water for 15 mins and eosin solvent for 30 secs. The slices were sequentially placed in 70%, 80%, 90% ethanol for 3 mins, correspondingly, and then 100% ethanol for 5 minutes. After that, they were put in xylene I and II for 5 minutes, respectively. Lastly, they were sealed with neutral gum and baked in the oven of 65℃ for 2 hours. Their images can be then checked by a microscope.

2.8 Immunohistochemistry Staining

Paraffin sections were routinely dewaxed with sodium citrate buffer in a high-pressure cooker for 15 mins. After the high-pressure cooker recovered in normal pressure, the slices were washed by PBS for three times and 5mins each time. They were then incubated in 3% H₂O₂ blocking solution to inhibit non-specific staining caused by endogenous peroxidase. Firstly, the slices were incubated in 10% sheep serum for 10 mins with primary rabbit anti-Ionized calcium-binding adapter molecule 1 (IBA1; 1:2000; Abcam; UK) and mouse anti-glial fibrillary acidic protein (GFAP; 1:2000; Abcam; UK), at 37°C for 2 hours. Then the slices were washed by PBS for three times and 5mins each time. Secondly, the slices were incubated with goat anti-rabbit secondary antibody (1:100; Zhongshan Jinqiao; Beijing) at 37°C for 30 mins. They were then washed by PBS for three times and 5mins each time. The colors come out under microscope after the slices were adding DAB (Zhongshan Jinqiao; Beijing) for staining 5 mins. They were then washed by water and put with hematoxylin and eosin for 3 mins and washed by water for 5 secs. Following that, they were put sequentially placed in 70%, 80%, 90% ethanol for 3 mins, correspondingly, 100% ethanol for 5 minutes, and then xylene I and II for 5 minutes, respectively. Finally, they were sealed with neutral resin and ready for imaging under microscope.

2.9 Image analyses

The immunostained slices obtained from the cortex and hippocampus were observed using OLYMPUS positive fluorescence microscope with 400x magnification (about 1mm*0.5mm in size). We used the built-in Color Deconvolution (H-DAB) function of Fuji’ image analysis software to deconvolve images to reduce the size of picture. Finally, the area of the positive region for IBA1 and GFAP divided by the total organization area will be reported.

2.10 Statistical analysis

The statistical analysis was performed using IBM SPSS Statistics version 23.0 and GraphPad Prism 8.0.2 version. All values in the experiment were expressed as mean ± SEM. The t-test was used for comparing two groups of data following normal distribution, and one-way ANOVA was adopted to compare more than three groups of data with normal distribution. The Wilcoxon test was used for data not following normal distribution. The Tukey's Honestly Significant Difference method was used for post-hoc multiple test. A p-value < 0.05 comparison was considered significantly different.

Table 1

The survival rate of rats on 8th and 21st postnatal day
	Time	sham	HIBD	MSC1	MSC2	MSC3	MSC4
Death/total(n)	P8	0/18	5/27	0/21	0/22	0/20	0/21	ns.
	P21	0/18	3/24	4/17	5/17	3/17	6/15	ns.

From Table 1, there was no difference in mortality between various groups on the 8th postnatal day and the 21st postnatal day. In total, of the134 rats, there are 26 rats died: five rats died either about 10 minutes after anesthesia induction or about 5 minutes after carotid artery ligation in the HIBD group; 21 rats in different groups died about 14 days after birth, which indicates that the death of rats maybe not directly due to injection of stem cells, rather because of HIBD modeling.

3.1 Human umbilical cord mesenchymal stem cells could obviously alleviate brain tissue damage.

As shown in Fig. 1A, at postnatal day 7 (P7), rat underwent permanent right common carotid artery occlusion. Three days after the injury (P10), they were administered vehicle or umbilical cord-derived mesenchymal stem cells (UC-MSCs) intranasally. On day 10 and 21, hanging wire test were conducted. Morris water maze tests were performed at P21–P26. After sacrifice, morphological and immunohistopathological assessments were conducted. As shown in Fig. 1B, hypoxic-ischemic modeling could obviously infarct the injured brain tissue, and the infarct area could be significantly reduced after the treatment of human umbilical cord mesenchymal stem cells.

3.2 Comparison of Different Doses of hUC-MSCs on Learning and Memory Ability improvement of HIBD rats

The Morris Water Maze test was used to assess learning and memory ability of rats from the 21st postnatal day and last for six days. On the 1st day, the difference of groups of the swimming speed and the escape latency was not statistically significant (P > 0.05, Fig. 2A). From the 2nd to the 5th day, each group’s escape latency was gradually shortened. No doubt that the escape latency was significantly longer in the HIBD group than in the sham group (P < 0. 01, Fig. 2A). All the four MSC groups had significantly shorter escape latency compared to the HIBD group (P < 0.01, Fig. 2A). On the 6th day, compared with the sham group, the time spent in the platform quadrant of the HIBD group was remarkably reduced (P < 0.001, Fig. 2B). In contrast with the HIBD group, the four MSC groups all stayed longer in the platform and crossed the platform more frequently (P < 0.01, Fig. 2B). These results indicate that the four doses of MSCs all significantly improved the learning and memory ability of HIBD rats.

Among the four MSC groups, the MSC3 group showed the shortest time to find the platform (P < 0.01, Fig. 2A). Besides, according to the trajectories of rats in each group searching for the platform in Fig. 2C, initially, they are generally random and marginal, as training duration increased, purposeful and trend-oriented movement trajectories were observed in both Sham and MSC groups, while HIBD groups remains random and marginal until the end of training. Among the four MSC groups, MSC3 group performed best on learning and memory ability.

3.3 Comparing different doses of hUC-MSCs on motor function improvement of HIBD rats

The hanging wire test results showed that each group hung longer on day 21 than on day 10 (Fig. 3C). The HIBD group had a significantly shorter hanging time than the sham group (P < 0.01; Fig. 3A, B, C). Compared to the HIBD group, all the four MSCs groups hanged longer (Fig. 3C). These results showed that stem cell therapy improved front foot motor function, and MSC3 performs better than MSC1 and MSC2, and comparable with MSC4 (Fig. 3C).

3.4 Comparing different doses of hUC-MSCs on body weight gain of HIBD rats

From Fig. 4, the body weight gain of the HIBD group was significantly lower than the sham group administered 0.9% normal saline(vehicle) on both the 21st and 28th postnatal day (P < 0.001; Fig. 4A, B). On the 21st day, MSC3 improved the weight gain better than MSC1 and MSC2, and comparable with MSC4 (Fig. 4A). On the 28th postnatal day, the rats’ gain was increased as the dose of stem cell therapy increased (Fig. 4B). These results indicate that stem cell therapy improve body weight gain in HIBD rats, MSC3 performed better than the lower doses and comparable with MSC4 on 21st postnatal day; while on the 28th postnatal day, the more dose of stem cells, the more improvement.

3.5 Treatment with MSC mitigated HIBD morphological changes in rat cerebral cortex and hippocampus CA1 area of HE staining

HE staining was utilized to observe the morphological changes of rat brains in each group. Generally, MSC groups significantly recovered the brain tissue injury compared with the HIBD group. The morphology of the cerebral cortex of rats in the sham group showed that the neuronal cells are large and full– the nucleus was large, the staining and the nucleoli were clear (Fig. 5). In the HIBD group, white bright areas (red arrows) representing cell edema were visible around the nucleus of neurons and lots of nuclei degeneration (yellow arrows) in neuronal cells were observed (Fig. 5). In four MSCs groups, neuronal cell edema and nucleus deformation were also observed, but these changes were milder than HIBD group (Fig. 5). These results indicate that MSCs can recover some brain tissue injury from HIBD.

Cerebral ischemia and hypoxia cause delayed-type cellular apoptosis, resulting in neuronal loss in hippocampal CA1 region and subsequently lead to a decline in cognitive function. We thus selected the hippocampus CA1 region to investigate neuronal injury caused by ischemia and hypoxia. It showed a significant loss of neurons, accompanied by shrinkage and loose arrangement in the hippocampus CA1 areas in the HIBD group compared to the sham group. Furthermore, there was an absence of normal cell structure, and deep staining was observed in the inner core area. The MSC groups significantly reduced the loss of neurons in cerebral cortex and CA1 of hippocampus compared with HIBD group. Among the MSC groups, MSC2, MSC3, MSC4 groups performed comparably, and they all performed better than MSC1 group(Fig. 5).

3.6 Immunohistochemistry of glial cells in cortex and hippocampus of the brain

The microglia activation reflects the degree of inflammatory cell infiltration. We used The proportion of positive area of IBA1 is used to represent how much microglia were activated. The area ratio and number of IBA1-positive regions in the cerebral cortex and hippocampus of rats on day 28 were shown in Fig. 6.

In cortex, HIBD group showed significantly larger microglia cell bodies and IBA1 expression area than the sham group (Fig. 6). The four MSC groups, except MSC2 group, significantly reduced the expression area of IBA1 compared to the HIBD group. Among them, MSC3 and MSC1 performed comparably better than other two groups.

In terms of the number of microglia, HIBD group showed significantly more than sham group. The four MSC groups performed comparably, and they all significant reduced the number of microglia compared to the HIBD group (Fig. 6).

In hippocampal CA1 and CA3 region, similarly to the cortex, in terms of IBA1-positive area, HIBD group showed significantly bigger than the sham group, the four MSC groups significantly reduced it compared with HIBD group, and MSC3 performed best in the four MSC groups (Fig. 6). In terms of the number of microglia, HIBD group activated more than the sham, the four MSC groups significantly reduced it compared with HIBD group in both hippocampal CA1 and CA3 region. Among the four MSC groups, no significant differences on CA1 region, while MSC4 performed best, but comparably with MSC3 group in CA3 region (Fig. 6).

We also compared the astrocytes cells by immunohistochemistry to GFAP on hippocampus and cortex of rats on day 28 as shown in Fig. 7. It showed that HIBD group had significantly more GFAP-positive area and the number of GFAP than the sham group in both hippocampus CA1 and cortex region. In terms of GFAP-positive area, the four MSC groups all significantly decreased compared to HIBD group, and no statistically significant difference among the four MSC groups. Regards to the number of GFAP, the four MSC groups were all significantly lower than the HIBD group. Among them, MSC2 and MSC3 performed comparably and better than the other two in cortex, while MSC3 performed best in hippocampal CA1 region. Overall, MSC3 performed best in hippocampal CA1 and cortex region (Fig. 7).

Hypoxic ischemic brain injury causes permanent damage to movement, sensation, perception, cognition, communication, behavior, and musculoskeletal problems in children. The effective treatment to recover the HIBD remains open. The mild hypothermia therapy provides neuroprotection in children with hypoxic-ischemic encephalopathy, but the improvement in prognosis is not significant.

Recent studies demonstrates that hUC-MSCs therapy was effective on treatment of HIBD. Mesenchymal stem cells have low immunogenicity to be transplanted through autologous or allogeneic transplantation applications for treatments of a variety of diseases(Weng et al. 2022). For example, MSC performed as a potential treatment to prevent complications in association with acute graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (Brooks et al. 2022). Due to the safety of MSCs, we did not use immunosuppressants in this study.

Previous studies showed that the mechanisms of MSCs’ neuroprotective effect on hypoxic-ischemic injury included inhibiting apoptosis of nerve cells, migrating to the site of brain injury, reducing the volume of infarction, and inhibiting secretion of inflammatory factors(Li et al. 2020b). Many animal experiments also showed that MSCs transplantation can restore motor function and improve memory in rats. A study(Zhang et al. 2019) showed that transplantation of MSCs after hypoxia ischemia can reduce the number of astrocytes, the loss of striatal neurons and significantly improving brain function in rats during recovery. It has been shown that MSCs can migrate to the infarct site of hypoxic-ischemic brain injury, differentiate into neurons or Schwann cells through paracrine effects, and promote nerve regeneration to function(Cai et al. 2017; Li et al. 2020b; Heris et al. 2022). The beneficial effect of stem cell therapy for brain injury models in rat function recovery and survival had been demonstrated in various animal studies(Sun and Kurtzberg 2021; Muhammad et al. 2022). Neuroprotection, neovascularization, neuronal regeneration, microglial pyroptosis and ferroptosis had all been proved after cell transplantation in animal models(van Velthoven et al. 2010; Xing et al. 2014; Huang and Zhang 2019; Hu et al. 2021; Zhai et al. 2022).

At present, stem cell therapy for cerebral ischemia and hypoxia has been applied in clinical trials and has shown good efficacy(Luo et al. 2023). However, the best practice of hUC-MSCs therapy remains open. The controversial issues such as optimal cell type, dose, timing, route of administration, and other variables still need to be refined for clinical treatment.

In our study, we investigated the best dose of hUC-MSCs for HIBD treatment by comparing four doses, 0.5* 10⁶ (MSC1), 1.0* 10⁶ (MSC2), 1.5* 10⁶ (MSC3), and 2* 10⁶ (MSC4) group.

In an earlier study(Tanaka et al. 2018), two different doses of HUC-MSCs in neonatal stroke mice showed that the high-dose(1*10⁵) HUC-MSC treatment was significantly decreased compared with the vehicle treatment on IBA1 positive staining area of cortex. In addition, Donega et al. 2013 have explored the adequacy of different doses of stem cells in the cure of cerebral palsy rat. The study used three different doses of stem cells (0.25*10⁶, 0.5*10⁶, 1*10⁶) to inject through the nose. They showed that both the 0.5*10⁶ and 1*10⁶ MSCs groups significantly improved the motor function of rats, while the low-dose 0.25*10⁶ group had no significant effect, and increasing the dose to 1.0×10⁶ did not further improve sensorimotor function or reduce cerebral infarction area. Therefore, they concluded that at least 0.5*10⁶ mesenchymal stem cells are required in the HI-induced brain injury model to have a long-term effect on functional recovery and infarct size.

Intranasal administration is non-invasive thus can protect rat from invasive risks, and previous studies showed that intranasal injection is safe and effective, we chose the nasal route. The success of MSC transplantation is also affected by the time of transplantation. The blood-brain barrier (BBB) may lead to the failure of stem cell transplantation. Hypoxic-ischemic injury can cause the opening of the blood-brain barrier, which consecutively leads to a series of secondary pathologies. Early BBB opening occurs within hours of ischemia and may close by itself(Ek et al. 2015; Hatayama et al. 2022; Park et al. 2023). The second stage of BBB begins 24–72 hours after ischemic injury(Lehner et al. 2011; Abbasloo et al. 2023). In short, early BBB correspondence is damage to tight junction function; while late BBB opening may be associated with neuroinflammation and disruption of function(Ek et al. 2015; Park et al. 2023). The mechanisms of BBB include recruiting immune cells(Mei et al. 2021), regulating metalloproteinases(Lan et al. 2019), maintaining morphological stability(Gussenhoven et al. 2019), and the interaction of various cellular components of the blood-brain barrier(Tjakra et al. 2019). As MSCs can only reach the site of injury for a short time, transplantation too early or too late may not be effective. We used the 72-hour time point in our study.

Although there are many experiments that have proved that mesenchymal stem cells are effective in treating rats with hypoxic-ischemic brain injury. However, few studies have been able to compare the effects of treatments between different dose groups of stem cells. Therefore, we designed behavioral experiments and brain pathology sections to assess motor and learning capacity, as well as pathological manifestations. In addition, our study confirmed the efficacy of four different doses of mesenchymal stem cells in rats with hypoxic-ischemic brain injury.

The Morris Water Maze test showed that the MSC3 (1.5*10⁶) group performed best, with the shortest time to find the platform, suggesting that it was the best on learning and memory function. In addition, the hanging wire test compared the motor function of rats’ forearm muscles, which also showed MSC3 performed best on recovering motor function.

Inflammation is one of the main pathogenic manifestations of hypoxic-ischemic brain injury, and microglia mainly activate the inflammatory response(Li et al. 2020a). Microglia are widely distributed in the brain and acted as an immune role in injury(Li et al. 2020a; Shao et al. 2021). In response to various stimuli, such as inflammation, hypoxia, ischemia and tumors, peroxiredoxin released extracellular activates Toll-like receptors and induces the production of inflammatory cytokines such as Interleukin-1β, IL-23, and Tumor necrosis factor α(Mika et al. 2013; Hickman et al. 2018; Donnelly et al. 2020). Moreover, it has been shown that microglia are more prevalent in the cerebral cortex and hippocampus than in the midbrain and hypothalamus(Fleiss et al. 2021). We thus chose the cortex and hippocampus to compare the microglia activation in different groups. The widely used microglial marker, ionized calcium-binding adapter molecule 1 (IBA1), was also used for comparison of neuropathologically-standard hypoxic ischemic injury. It showed that the IBA1-positive region of all MSC groups (MSC3 group performed best) were significantly smaller than the HIBD group in hippocampus CA1, CA3 and cortex. This suggests that the underlying mechanism of mesenchymal stem cell therapy for hypoxic-ischemic brain injury may be related to reducing the microglial activation.

Astrocytes are the largest class of glial cells in the mammalian brain and play an important neuroprotective role in cerebral ischemic injury (Jiao et al. 2020). A well-known astrocytes marker, GFAP was used to detect the morphological changes and number of astrocytes (Hol and Pekny 2015). It has been shown that the reduction of early astrocytes was closely related to clinical symptoms(Colombo and Farina 2016); in chronic central nervous system diseases, astrocytes can produce lactose ceramide, promote inflammatory response and lead to neurodegeneration(Mayo et al. 2014). These results suggest that astrocytes are two-sided effects, depending on the timing of action, specific diseases, and different stimuli of the microenvironment. Our results showed that astrocyte activation (more GFAP positive expression) was truly boosted in the HIBD group compared with the sham group, and all the MSC groups performed comparably on reducing the astrocyte activation of HIBD. It also indicated that the mechanism of MSC in the treatment of cerebral ischemia and hypoxia may be closely associated with the activation of astrocytes.

However, there are still some limitations in our study. First, although we use the well-known modified mice method for hypoxic-ischemic brain damage, this method, like all animal models, does not fully simulate human disease. Alternative methods, such as bilateral common carotid artery occlusion, are known to produce significant hypoxic-ischemic brain injury in both location and magnitude. Therefore, it is necessary to validate the results of our study using other types of HIBD animal models in newborn rats. Secondly, to achieve the best clinical treatment efficacy, problems such as route of administration, timing of administration, injection rate, and transplantation frequency still need to be addressed. Therefore, there is a long way to go to translating animal experiments into clinical treatments.

Our results show that MSC exerts neuroprotective effects in the HIBD experimental rat model. The activation of microglia and astrocytes were observed in HIBD group, and they were reduced significantly by MSCs, indicating that they were related to the underlying mechanisms of MSC treatment of HIBD. Comparing four doses of MSC groups, they generally significantly increases the body weight, and restores the learning and memory function, reduce the microglial and astrocytes activation of HIBD rats. Among them, MSC3 group performed best on improving body weight gain, learning and memory ability of HIBD rats. In addition, high dose MSC groups (MSC3, MSC4) significantly reduced microglial activation, and MSC3 significantly reduced astrocytes activation in the HIBD rats. Overall, MSC3 (1.5* 10⁶) should be the best dose for HIBD treatment. Our research demonstrated that all the four doses of MSC were effective in the HIBD rats. In a certain range, high dose of stem cells can improve the learning and memory function and neuroprotective effect of rats. The optimal MSC dose provides experimental animal basis for guidance of clinical use.

Ethical Approval

All the animal experiments are approved by the ethical review of animal welfare in Hubei University of Medicine.

Competing Interests

The authors declare no conflict of interest.

Funding

This study was supported by the Science and Technology Program of Hubei Province, China (No.2013BCB002, NO.2021CFB158), Key Research Project of Hubei Provincial Department of Education (No. D20222106) and National Natural Science Foundation of China (No. 32060150).

Authors Contributions

Mengxue Yang conducted the experiments and wrote the initial draft, Kun Zheng conduct some experiments and revised the draft, Ruibo Zhang, Guangzhen He, Jinyun Xu, Hao Jiang, Lan Ren, and Liang Zhao contributed on experiment design and draft revision. Yan Liao and Zeqin Fu contributed on human umbilical cord mesenchymal stem cells culture. Wenting Liu and Jiaowei Gu supervised the whole project.

Availability of data and materials

The data and materials are available for academic use upon request.

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The optimal dose of human umbilical-cord mesenchymal stem cells (hUC-MSCs) on hypoxic ischemic brain damage(HIBD) rats via intranasal transplantation

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Abstract

Figures

1 Introduction

2 Materials and methods

2.1 Animals and the HIBD Model

2.2 Human Umbilical Cord Mesenchymal Stem Cells (hUC-MSCs)

2.3 Intranasal Injection of hUC-MSCs

2.4 Hanging Wire Test

2.5 Morris Water Maze

2.6 Body Weight Measurement

2.7 Hematoxylin-Eosin Staining

2.8 Immunohistochemistry Staining

2.9 Image analyses

2.10 Statistical analysis

3 Results

3.1 Human umbilical cord mesenchymal stem cells could obviously alleviate brain tissue damage.

3.2 Comparison of Different Doses of hUC-MSCs on Learning and Memory Ability improvement of HIBD rats

3.3 Comparing different doses of hUC-MSCs on motor function improvement of HIBD rats

3.4 Comparing different doses of hUC-MSCs on body weight gain of HIBD rats

3.6 Immunohistochemistry of glial cells in cortex and hippocampus of the brain

4. Discussion

5. Conclusion

Declarations

Ethical Approval

Competing Interests

Funding

Authors Contributions

Availability of data and materials

References

Supplementary Files

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