In spite of its known toxic effects on the cerebellum, monosodium glutamate (MSG), is widely used in food industry in our world today. The representing symptom for its toxic effects is ataxia [1]. This debilitating disease is caused by sustained high concentrations of MSG, as an excitatory amino acid, in the synaptic cleft region resulting in excessive glutamate receptor activation with persistent depolarization producing metabolic and functional exhaustion of the affected neurons leading to neural necrosis [2]. Moreover, the increased levels of glutamate lead to increased calcium entry, internal oxidative stress with generation of free radicals, mitochondrial dysfunction, and eventually necrosis [3]. It was found that human umbilical cord blood stem cells (CD34+/ CD34-) or human bone marrow, although hematopoietic in nature, under certain conditions can change their natural fate and display neural features [4], so they can be good candidates for treating spinocerebellar ataxia. However, no studies have compared the efficacy of the different types of cells. Thus, the purpose of this study is to compare the effect of transplantation of human umbilical cord blood stem cells; CD34+ versus CD34-, on cerebellar damage and motor function deterioration induced by monosodium glutamate in a preclinical animal model.

Animal model

Forty adult female albino rats weighing 120-150 grams were used in this study, divided into 4 groups. Group I (Control group): animals in this group received daily intraperitoneal (IP) injection of one ml distilled water for 10 days followed by a single dose of one ml intravenous (IV) injection of distilled water on the 11^th day. Group II (MSG group): animals in this group were subjected to chemically induced cerebellar injury by daily IP injection of 6 mg/Kg of MSG for 10 days [5]. Group III (CD34+Stem cell group):Cerebellar damage was done using MSG for 10 days as in the previous group followed by single intravenous injection , in the tail vein, of 106 CD34+ stem cells on the 11^th day. Group IV (CD34-Stem cell group): The same as group III except using 106 CD34- stem cells on the 11^th day instead of 106 CD34+ stem cells. All animals were sacrificed by decapitation under light anesthesia after 4 weeks of intravenous injection of different treatments.

Isolation and culture of stem cells

Both CD34+ versus CD34- stem cells of umbilical cord blood were separated by Ficoll-Hypaque method followed by immunomagnetic separation of CD34+ stem cells [5]. CD34- stem cells have capacity of adhesion to the flasks, as opposed to other types of cells in the CD34 negative fraction, which were eliminated from the culture during the procedures of medium change [6-8].

Histological, immunohistochemical examination

6μm cerebellar paraffin sections were stained by Haematoxylin and Eosin (H, E), Mallory's phospho tungestic acid haematoxylin (PTAH), cresyl fast violet and immunohistochemical stain using polyclonal rabbit anti-glial fibrillary acidic protein (anti-GFAP). The GFAP stain is specific for the intermediate filament fibrillary acidic protein found in astrocytes.

Qualitative and quantitative assessments were done for histological, immunohistochemical changes in the cerebellum. Quantitative assessment included measurement of neuronal density [9], linear density of Purkinje cells [10], density of stellate and basket cells, density of granule cells and mean dendrite length. Optical density was done for Nissl’s granules and astrocytic GFAP activity.

Detection of Sry gene

Because we collected umbilical cord blood of male fetuses, PCR analysis of male-specific Sry gene was done to detect human male Sry gene sequence in the cerebellum of the female rats.

Accelerating rotarod test

Motor assessment was done by Accelerating Rotarod Test on the rotarod [7]. Animals underwent linear acceleration from 4 to 40 rpm in 300 seconds. Latency to fall from rotarod was recorded in seconds. Each trial lasted for a maximum of 5 min and rats were rested for a minimum of 15 min between trials to avoid fatigue. After the rotarod test, the body weights of rats were recorded. Rats underwent three trials per day for four consecutive days, and the mean latency to fall on each day was recorded for statistical analysis.

Beam balance test [8]

Motor coordination was also assessed using a beam balance test. This test essentially examines the ability of the animal to remain upright and to walk on an elevated and relatively narrow beam. Score from 0 to 5 was used as following: 5: Walks the balance beam flawlessly, does not need to check balance, does not pause, and completes the walk within six seconds. 4: Walks the beam, but is somewhat unsteady, completes the walk within six seconds. 3: Walks the beam, but is somewhat unsteady, may pause one or more times, takes more than six seconds to complete the walk. 2 : Walks the beam, but is very unsteady, almost falling off, may pause one or more times, and/or takes more than six seconds. 1: Falls off the beam before completing the walk. 0: Falls off the beam immediately.

Statistical analysis

Data are presented as the mean ± standard deviation (SD). The results were compared using Student T test for the histological changes and one-way ANOVA for physiological tests. Statistical significance was determined at 95% confidence interval.

Histological changes

Group II (Monosodium Glutamate group) -: In this group, the mean number of cells in the molecular layer was decreased (39.38 ± 6.4 cells / HPF) (Figure 5) and the mean number of Purkinje cells was also decreased (11 ± 2.4 cells/HPF) (Figure 6). Purkinje cells showed complete loss (10%), shrinking leaving vacuoles in intercellular spaces (40%) and necrosis (30%) (Table 1) (Figure 1b compared to Figure 1a). Shrunken neurons (80%) were detected in the deep cerebellar nuclei. Also, granule cells appeared less crowded with mean density of 119.1 ± 0.2987 cells /HPF compared to control group (Figure 7). The mean optical density of Nissl’s granules in the perikarya of Purkinje cells was markedly decreased (0.1056 ± 0.050) (Figure 8) (Figure 2b compared to Figure 2a)) and the mean length of the dendrites was also markedly decreased (17.85 ± 0.015) compared to control group (Figure 9) (Figure 3b compared to Figure 3a). Marked increase of GFAP immunoexpression was noticed in the cytoplasm of many astrocyte cell bodies and processes in the granular layer in all animals of this group compared to the positive staining of the control group. (Figure 4b compared to Figure 4a). The mean optical density of GFAP activity in astrocytes of this group was 0.51 ± 0.056 which was significantly higher than that of the control group (Figure 10).

Figure 1: (1a): A photomicrograph of a section in the cerebellar cortex of a rat from control group showing scattered small stellate (Sc) and basket cells (B) in the molecular layer. Large pyriform cells with vesicular nuclei (P) are shown in the Purkinje cell layer. Crowded small cells with deeply stained nuclei (G) are shown in the granular layer [H&E x 400]. (1b): A photomicrograph of a section in the cerebellum of a rat from group II showing decreased density of neurons in the three cortical layers. Purkinje cells (P) are widely displaced and distorted. Granule cells (G) appear less crowded than in the control group [H&E x 400]; (1c): A photomicrograph of a section in the cerebellum of a rat treated with CD 34+ stem cells showing restoration of the normal architecture of the cortex,similar to that of the control group. There is slight increase in the density of the neurons in the three cortical layers [H&E x 400].(1d): A photomicrograph of a section in the cerebellum of a rat treated with CD 34- stem cells showing increased number of Purkinje cells and restoration of the normal architecture of the cortex,similar to that of the control group [H&E x 400].

Figure 2: (2a): A photomicrograph of a section in the cerebellar cortex of a rat from control group showing purple Nissl granules (arrow) in the perikarya of Purkinje cells [Cresyl fast violet x 1000]. (2b): A photomicrograph of a section in the cerebellar cortex of a rat from group 2 showing decreased purple Nissl granules (arrow) in the perikarya of Purkinje cells [ Cresyl fast violet x 1000]. (2c): A photomicrograph of a section in the cerebellar cortex of a rat treated with CD 34+ stem cells showing increase in purple Nissl granules in the perikarya of Purkinje cells, compared to that of MSG group [Cresyl fast violet x 1000].(2d): A photomicrograph of a section in the cerebellar cortex of a rat treated with CD 34-stem cells showing increase in purple Nissl granules ( arrow) in the perikarya of Purkinje cells,compared to that of MSG group [Cresyl fast violet x 1000].

Figure 3: (3a): A photomicrograph of a section in the cerebellar cortex of a rat from control group showing delicate dendritic arborizations of Purkinje cells extending up into molecular layer [PTAH x 400].(3b): A photomicrograph of a section in the cerebellar cortex of a rat from group II showing decreased length of Purkinje cell dendrites [PTAH x 400].(3c): A photomicrograph of a section in the cerebellar cortex of a rat treated with CD 34+ stem cells showing increase in the length of dendrites of Purkinje cells [PTAH stainX400]. (3d): A photomicrograph of a section in the cerebellar cortex of a rat treated with CD 34- stem cells showing long dendrites of Purkinje cells extending into the molecular layer [PTAH stainX400].

Figure 4: (4a): A photomicrograph in the cerebellum of control rat showing positive brownish immunoreaction in some cells of the granular layer (astrocytes) [GFAP x 1000]. (4b): A photomicrograph of a section in the cerebellum of a rat from group II showing increase in staining intensity and number of positive brownish star shaped cells in granular layer (astrocytes)[GFAP x 1000]. (4c): A photomicrograph of a section in the cerebellum of a rat treated with CD 34+ stem cells showing increase in staining intensity and number of positive brownish star shaped cells in granular layer (astrocytes),compared to that of control group but less than group II [GFAP x 1000] (4d): A photomicrograph of a section in the cerebellum of a rat treated with CD 34- stem cells showing increase in staining intensity and number of positive brownish star shaped cells in granular layer (astrocytes),compared to that of control group but less than that of group II , 3 [GFAP x 1000].

Figure 5: The mean and standard deviation (SD) of the density of molecular layer cells in the different experimental groups.*Statistically significant compared to control group: P<0.05 **Statistically significant compared to group II: P<0.05.

Figure 6: The mean linear density of Purkinje cells/ mm length of the folia in the different experimental groups. *Statistically significant compared to control group: P<0.05 **Statistically significant compared to group II: P<0.05.

Figure 7: The mean and standard deviation (SD) of the density of granule cells of granular layer in the different experimental groups. *Statistically significant compared to control group: P<0.05 **Statistically significant compared to group II: P<0.05.

Figure 8: The mean optical density of Nissl’s granules in the different experimental groups.*Statistically significant compared to control group: P<0.05**Statistically significant compared to group II: P<0.05.

Figure 9: The mean dendrite length in the different experimental groups.*Statistically significant compared to control group: P<0.05**Statistically significant compared to group II: P<0.05.

Figure 10: The mean optical density of GFAP activity in the different experimental groups.*Statistically significant compared to control group: P<0.05**Statistically significant compared to group II: P<0.05.

Group III (CD34+Stem cells given after IP injection of monosodium glutamate): The mean number of cells in the molecular layer was significantly increased in this group (44.67 ± 3.3 cells /HPF) compared to group II (Figure 5). Most of the Purkinje cells (60%) were shrunken with some degenerative changes. Only 15% of Purkinje cells showed complete loss (Table 1). The mean number of cells in the Purkinje cell layer was higher (13.667 ± 8.5 cells/HPF) than in group II, but it was still less than that of the control group (Figure 6). Shrunken multipolar neurons (40%) were seen in the region of deep cerebellar nuclei. Also, granule cells were increased to 173.2 ± 0.2027 compared to group II (Figure 7) (Figure 1c).The mean optical density of Nissl’s granules in the perikarya of Purkinje cells was increased (0.2057 ± 0.043) (Figure 8) (Figure 2c) .The mean length of the dendrites was increased (30.82 ± 0.012) (Figure 9) (Figure 3c) compared to group II but was less than that of the control group. Increased staining of the many soma and processes of astrocytes compared to control group was seen (Figure 4c). The mean optical density of GFAP activity in astrocytes of this group was 0.48 ± 0.051, which was significantly less than that of MSG group (Figure 10).

Table 1: The frequency distribution of the histopathological changes in Purkinje cell layer in the different experimental groups.

Group IV (CD34- Stem cells given after IP injection of monosodium glutamate): The mean number of cells in the molecular layer was increased in this group (60.5 ± 5.3 cells /HPF), compared to group II (Figure 5). Shrunken Purkinje cells with degenerative changes (40%) and complete cell loss of others (12%) were seen (Table 1). The mean number of Purkinje cells was increased to 17.5 ± 10.5 cells/ HPF (Figure 6). Shrunken multipolar neurons (30%) were seen in the region of deep cerebellar nuclei. Granule cells were increased to 177 ± 0.2027 cells/HPF compared to group II (Figure 7) (Figure 1d).The mean optical density of Nissl’s granules in the perikarya of Purkinje cells was markedly increased (0.2654 ± 0.042) (Figure 8) (Figure 2d). The mean length of the dendrites in this group was increased (34.53 ± 0.013) compared to the control group (Figure 9) (Figure 3d). There was an increase in the staining of soma and processes of astrocytes compared to the control group (Figure 4d). The mean optical density of GFAP activity in astrocytes of this group was 0.36 ± 0.054, which is significantly less than that of the MSG and CD34+ groups (Figure 10).

The PCR product could not detect Sry gene in neither the CD34+ nor the CD34- groups. Therefore, 100% of cerebellar specimens of female recipient rats were negative for the presence of SRY gene.

Accelerating rotarod test

Group II showed gradual deterioration in motor function throughout the time of the experiment compared to control group. Groups III and IV showed gradual deterioration through the first 2 weeks of assessment followed by some improvement along the remaining weeks, but did not return to baseline values (Figure 11).

Figure 11: Results of accelerating rotarod testp˂0.05.

Balance beam test

Cerebellar injury resulted in motor function incoordination which was assessed according to the scores shown in Table 2. Group I (control): the rats walked freely and achieved an average score of 5, while group II (MSG) rats were unsteady and took longer times to finish the walk, thus achieving an average score of 3, indicating gradual deterioration of the motor function compared to control group. Group III (CD34+) showed some improvement with an average score of 4. Group IV (CD34-) also showed similar improvement and a similar score of 4 as group (CD34+).

Table 2: The average scoring of balance beam test in the different experimental groups: P < 0.05.

Motor function of rats was evaluated by an accelerating protocol of rotarod as well as by beam balance test throughout the 4 weeks of the study period. Gradual improvement in motor function could be detected in the 2 groups injected by stem cells. This improvement might be explained by the formation of new growth factors or activation of endogenous stem cells. Better results were obtained in other studies with larger numbers of animals, other routes of administration of stem cells (intracranial), and longer periods of study [11].

Jonathan et al. 2010 have shown that mesenchymal stem cells are capable of integrating into the central nervous system, migrate towards the areas where neurodegenerative processes are occurring, and rescue the degenerating cells through cell trophic effects [12].

The MSG resulted in significant cerebellar damage. Necrosis of Purkinje cells, detected in all animals of this group, was the most evident pathological changes of this cerebellar damage. This was accompanied by decreased neuronal density in the molecular and granular layers. Similar results were shown by Hashem et al. 2012 [13] who found necrotic changes in Purkinje and also in granule cells in adult male albino rats receiving MSG orally (3 g/kg/day). Also, Eweka and Om 'Iniabohs 2007 [1] found decreased granule, Purkinje cell density in rats, fed orally by 6g MSG for 15 days. This necrotic effect of MSG may be due to sustained high concentrations of MSG, as an excitatory amino acid, in the synaptic cleft region resulting in excessive glutamate receptor activation with persistent depolarization producing metabolic and functional exhaustion of the affected neurons and hence leading to neural necrosis [2,14]. The neurotoxic effect of MSG could be mediated by an oxidative stress process resulting in the depletion of glutathione and acute concentration dependent efflux of ascorbate from the cells (major cellular antioxidants) leading to a form of cell injury called oxidative glutamate toxicity [15,16]. In MSG group, the detected decrease in Nissl’s granule contents can be attributed to the downregulation of mRNA expression leading to a decreased rate of intracellular protein synthesis, which in turn leads to decreased Nissl granules [17,18]. The decreased length of the Purkinje cell dendrites noticed with MSG can be explained by Chen et al. 2003 [19] suggesting that MSG toxicity contributes to marked reduction of dendritic capacity of differentiation. The significant increase in the optical density of astrocytes in this group compared to the control group can be explained by the findings of Baydas et al. 2006 [20] and Sofroniew and Vinters 2010 [21], who reported that any mechanical, chemical or degenerative insults to the brain stimulate astrocyte proliferation and hypertrophy with increased synthesis of GFAP leading to vigorous astrogliosis.

Chang et al. 2011 showed that mesenchymal stem cell transplantation ameliorates motor function deterioration of spinocerebellar ataxia by rescuing cerebellar Purkinje cells [11], however degenerative changes and cell loss were still evident. GFAP immunostain showed a significant decrease in the optical density of astrocytes, compared to MSG group, denoting beginning of the healing process, however it was still higher than the control group. This is in accordance with the results of Ou et al. 2010 [22] who reported that after 28 days of transplantation of CD34+ cells (transfected with glial cell line-derived neurotrophic factor [GDNF] gene) in spontaneous hypertensive rats exposed to transient middle cerebral artery occlusion, there were still GFP positive astrocytes

CD34- stem cells treated group (MSG followed by CD34-) showed absence of necrotic changes; however degenerative changes in the form of shrunken cells were still seen. These cells may have the ability to reverse the injury and increase the number of normal cell, which could compensate for the loss of cells by recovering and restoring their cellular functions. The improvement induced by these CD34- stem cells is more marked than that induced by CD34+ stem cells. The percentage of degenerative changes is less, and there was significant increase in density of stellate, basket and granule cells compared to that of the CD34+ stem cell group. There was also an increase in both length of Purkinje cell dendrites and Nissl’s granule contents compared to CD34+ stem cell group.

No human DNA was found in the cerebellum of the CD34+ or CD34- treated groups in our study, which means that stem cell homing did not occur. Zietlow et al. 2008 [23] reported that host plasticity promotes modifying the diseased environment to stimulate the endogenous stem cells of the host brain by migratory stem cells to the areas of injury. One of the mechanisms that support our result was explained by Sipp 2011[24] who found that the paracrine secretion of cytokines secreted from CD34- stem cells induced neuroprotective, functional recovery and modulation of the immune response. Additionally, Koh et al. 2008 [25] detected that CD34- stem cell transplantation produces neurotrophic factors, including vascular endothelial growth factor, glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor, all of which are strong neuroprotectants. A significant decrease in the optical density of astrocytes was seen using GFAP immunostain, compared to CD34+ stem cell group; denoting that the healing process is more extensive and the damage is less than in the CD34+ stem cell group, denoting that restoration of cerebellar cell function was superior to the CD34+ stem cell treated group.

Intravenous infusion of HUCB stem cells, either CD34+ or CD34-, had the ability to recover the injured cerebellum and help its functional restoration. CD34- stem cells showed more improvements of cerebellar structure and function than CD34+ stem cells.

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