Intravenous Administration of Human Umbilical Tissue-Derived Cells Improves Neurological Function in Aged Rats after Embolic Stroke

Abstract

Intravenous administration of human umbilical tissue-derived cells (hUTC) improves neurological function in young adult rats after stroke. However, stroke is a major cause of death and disability in the aged population, with the majority of stroke patients 65 years and older. The present study investigated the effect of hUTC on aged rats after embolic stroke. Rats at the age of 18–20 months were subjected to embolic middle cerebral artery (MCA) occlusion. Two groups of eight animals each were compared. The investigational group was injected intravenously with 1 × 10⁷ cells/kg in serum-free culture medium (vehicle) 24 h after stroke onset, and the control group was treated with vehicle only at the same time poststroke. Intravenous administration of hUTC significantly improved neurological functional recovery without reducing infarct volume compared to vehicle-treated aged rats. Additionally, hUTC treatment significantly enhanced synaptogenesis and vessel density in the ischemic boundary zone (IBZ). Moreover, hUTC treatment resulted in a trend toward increased progenitor cell proliferation in the subventricular zone (SVZ) compared to vehicle-treated aged rats. Intravenous administration of hUTC improved functional recovery in aged rats after stroke. The enhancement of synaptogenesis and vessel density may contribute to the beneficial effects of hUTC in the treatment of stroke in the aged animal.

Keywords

Cerebral infarct Human umbilical cord Synaptogenesis Stroke recovery Aged rat model

Introduction

According to the 2011 statistical update on heart diseases and stroke in the US, approximately 795,000 people experience a new or recurrent stroke each year (50). Stroke is the third leading cause of death and a major cause of long-term disability in the US. The only therapeutic intervention available for stroke is the use of thrombolytic drugs, the success of which is governed by the narrow time window in which they need to be administered. Therefore, alternative strategies for stroke treatment are necessary. Infusion of cell-based therapies represents a promising approach for neurorestoration after stroke. Human cells isolated from various sources such as the brain, bone marrow, cord blood, and adipose tissue have been utilized for neurorestoration after stroke. Human umbilical cord blood cells (hUCBCs) are an attractive source of cells for cell-based therapies because they can be collected without the ethical conflicts that are associated with embryonic or fetal cells. Further, they have been shown to have the ability to develop into nonhematopoietic cells of various tissue lineages, including neural cells (21). In addition to cord blood, the umbilical cord tissue itself is a particularly attractive source of cells, especially considering that the cells can be harvested non-invasively from tissue that is normally discarded without the ethical conflicts of other cell sources (9,16,18,35,53). In fact, human umbilical tissue-derived cells (hUTC) have demonstrated a remarkable ability to improve neurological functional recovery in a rodent model of cerebral ischemia (58). The beneficial effects of hUTC are likely attributed to their ability to evoke host brain restorative processes including neurogenesis and vascular and synaptic plasticity (58). Previously, the effects of cord blood cells and hUTC in experimental stroke models have been exclusively studied in young adult rats in the age range of 12–13 weeks (10,48,56,58). However, none of the studies have focused on the therapeutic efficacy of umbilical cord blood- or tissue-derived cells to treat stroke in aged rats. In order to study the efficacy of treatment regimens in an age-associated disorder like stroke, it is crucial to use an age-appropriate animal model, as it has been demonstrated that aged animals respond differently to experimentally induced stroke than younger animals. For example, it has been shown that histopathological changes (the infiltration of macrophages into cerebral infarcts and the hypertrophy of astroglial fibrils surrounding these infarcts) induced by cerebral infarction were significantly reduced in aged animals (23–24 months old) when compared with young adult rats (2–4 months old; average rat life span being 27–29 months) (20). Although the mechanisms by which infused hUCBCs induce functional benefit after stroke are not clear (10), reduced inflammation and nerve fiber reorganization have been proposed as possible mechanisms of neurorestoration after embolic stroke (3,13,34). Since aging is associated with changes in the immune system, efficacy studies of therapeutic agents that participate in altering immune/inflammatory responses after cerebral infarction in young rats may not necessarily be extrapolated to old animals (3).

While the adult brain has the capacity for plasticity and self-repair following brain injury, brain plasticity postinjury declines with age (11,15,51,52). Clinical studies indicate that advanced age is a significant prognostic factor for poor stroke recovery (29,30,43), and stroke rehabilitation is less effective in the aged population compared with younger patients (46). In experimental stroke, previous studies indicate that aging is associated with diminished neurogenesis, angiogenesis, and synaptic plasticity, one or all of which can impair brain functional recovery after stroke (6,7,25,42,44,49). Therefore, while hUTC were shown to enhance motor-sensory recovery in young adult rats (58), no evidence existed to determine whether this effect would also be observed in aged animals. In the present study, we tested the effects of hUTC on neurological functional recovery in aged rats (18–20 months of age) when administered intravenously at 24 h after stroke. Our embolic stroke model in old rats is particularly important in light of the fact that nearly 75% of all strokes occur in people older than 65 years (37). Moreover, age is the single most important risk factor in stroke, and for each successive 10 years after age 55, the stroke rate more than doubles in both men and women (37). In contrast to the abundant studies of cell-based therapies in young adult rats, there is a scarcity of literature in aged mice. Our study is the first to describe the effects of hUTC in the aged rat after stroke.

Materials and Methods

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Henry Ford Hospital. All outcome measurements were assessed by observers blinded to the treatments.

Preparation of hUTC

Human umbilical tissue-derived cells (hUTC) were isolated from a single donor following informed consent and ethical approval as described elsewhere (35). Cell preparation for implantation was described elsewhere (58). Briefly, cells were prepared for injection immediately prior to delivery. Cell vials were thawed in a 37°C water bath, and then washed with vehicle, a serum-free proprietary formulation. Aliquots of the cell suspension were removed and mixed with trypan blue (Sigma, St. Louis, MO, USA), and cells were counted with a hemato cytometer. The cell concentration in the cell suspension was adjusted to the appropriate concentration with vehicle as determined by the animal weight (1 × 10⁷ cells/kg in 2 ml of vehicle). Vehicle-only samples were prepared for injection in control animals.

Experimental Protocol

Male Wistar rats of ages 18–20 months, weighing 450– 600 g (Charles River Breeding, France), are referred to as aged rats. All rats were subjected to embolic middle cerebral artery (MCA) occlusion (61). Briefly, rats were anesthetized with 3.5% isoflurane (Baxter, Deerfield, IL, USA) in N₂O/O₂ (2:1) and maintained at 1.5% isoflurane via a facemask. The right common carotid artery, external carotid artery, and internal carotid artery were exposed. A modified PE-50 catheter (BD, San Jose, CA, USA) with a 0.3-mm outer diameter filled with a single clot was gently advanced from the right external carotid artery (ECA) into the lumen of the internal carotid artery (ICA), near the origin of the MCA. The clot was then injected along with 2–3 μl of saline (61). To test the effect of hUTC in aged rats after stroke, hUTC at a dose of 1 × 10⁷ cells/kg (n = 8) were intravenously administered via the tail vein 24 h after stroke onset. The dose selected for the current study is based on our previous study, which demonstrated that intravenous administration of hUTC at a dose of 1 × 10⁷ cells/kg elicits neurorestorative responses in young rats after stroke (58). The same volume of vehicle only (2 ml) was administered to ischemic rats (n = 8) as a control group, at 24 h poststroke. For mitotic labeling of cell proliferation, bromodeoxyuridine (BrdU; Sigma) at a dose of 50 mg/kg was intraperitoneally administered to rats for 14 consecutive days, starting 24 h after stroke onset.

Functional Outcome

All functional outcome tests were performed weekly by observers blinded to the treatments, starting 1 day after onset of MCA occlusion.

Modified Neurological Severity Score (mNSS)

mNSS is a composite of motor, sensory, reflex, and balance tests (10). Neurological function was graded on a scale of 0 (normal) to 18 (maximal deficit).

Adhesive Removal Test

An adhesive removal test was applied to measure somatosensory function deficits after brain injury (54). Briefly, two pieces of adhesive-backed paper dots (113.1 mm²) were used as bilateral tactile stimuli occupying the distal-radial region on the wrist of each forelimb for the first 4 weeks after stroke onset. Thereafter, half-sized stimuli (56.6 mm²) were used to increase the difficulty of the adhesive removal test, starting 35 days poststroke. This method is sensitive in measuring long-term functional outcome after stroke (54). Each animal received three trials per testing day, and the mean time (seconds) required to remove stimuli was recorded.

Foot-Fault Test

Rats were tested for placement dysfunctions of forelimbs with the modified foot-fault test (23). The total number of steps (movement of each forelimb) that the rat used to cross the grid and the total number of foot-faults for the left forelimb were recorded. Data are presented as a percentage of left foot-faults.

Measurements of Infarct Volume

Rats were sacrificed 60 days after stroke. Infarct volume was measured on seven hematoxylin and eosin (H&E; Sigma)-stained coronal sections using a Global Lab Image analysis program (Data Translation, Marlboro, MA, USA), as previously described (61). The infarct volume is presented as a percentage of total contralateral hemisphere volume.

Immunohistochemistry

Immunohistochemical studies were performed on coronal sections corresponding to bregma −1.0 to 1.0 mm obtained from each experimental animal. To identify proliferating cells, monoclonal antibody (mAb) against BrdU (Dako, Carpinteria, CA, USA) was used at a titer of 1:100. For quantification, the number of BrdU-incorporating subventricular zone (SVZ) cells and the area of SVZ were measured. Data are presented as density of BrdU-immunoreactive cells within the SVZ.

To detect human cells, a mAb against human mitochondria (HM; Spring Bioscience, Pleasanton, CA, USA) was used at a titer of 1:100. The numbers of HM immunoreactive cells within the ipsilateral hemisphere were measured and presented as the density of HM-positive cells within the ipsilateral hemisphere. Double stainings were performed to visualize cellular colocalization of HM with the neuron-specific marker microtubule-associated protein (MAP2) (Aves, Tigard, OR, USA) at a dilution of 1:1,000, and with glial fibrillary acidic protein (GFAP; Dako), an astrocyte marker, at a dilution of 1:10,000.

For a morphological analysis of vessels, a polyclonal antibody against von Willebrand factor (vWF; Dako) was used at a titer of 1:400. The number of vWF-immunoreactive vessels in the ischemic boundary zone (IBZ) was measured, as previous described (58). The IBZ was defined as the morphological area surrounding the lesion cavity immediately adjacent to the nonischemic tissue with an approximately 500 μm wide frame. Data are presented as the density of vWF-immunoreactive vessels within the IBZ.

Synaptophysin is a glycoprotein found in presynaptic vesicles, which has been used as a marker for synaptic plasticity (36). To detect presynaptic plasticity and synaptogenesis, a mAb against synaptophysin (Chemicon, Billerica, MA, USA) was used at a titer of 1:500. The synaptophysin-immunoreactive area in the IBZ was measured. Data are presented as percentage of synaptophysin-immunoreactive areas within the IBZ.

Statistical Analyses

Behavioral data were evaluated for normality, and a nonparametric analysis was considered if data did not follow a normal distribution. An analysis of variance (ANOVA) was conducted to test the treatment effect on each outcome between groups at each time point, followed by the post hoc least significant difference tests. Histology data were evaluated using a Student t test. All data are presented as mean ± SE. A treatment effect was detected if p < 0.05.

Results

Mortality

The mortality rates were 33% (4 of 12) and 27% (3 of 11) for rats treated with hUTC and vehicle control, respectively. In addition, two animals died within 24 h after stroke onset prior to treatment. Autopsy revealed that these animals had massive ipsilateral hemispheric swelling and/or hemorrhage. No significant differences were detected among the groups. Rats that died were excluded from further evaluation. The total number of animals remaining in this study was eight per group.

Lesion Volume

Lesion volume was comparable between rats treated with hUTC (26.7 ± 7.3%) and vehicle (26.6 ± 9.4%).

Neurological Functional Outcome

All rats exhibited severe functional deficits 24 h after stroke onset and with no significant differences measured by mNSS, foot-fault test, and adhesive removal test among the groups. Administration of hUTC 24 h after MCA occlusion significantly (p < 0.05) improved neurological functional outcome as measured by the foot-fault test starting from day 35, and by the adhesive removal test starting from day 42, which persisted up to 60 days after stroke. In addition, rats treated with hUTC exhibited significant improvements in mNSS at day 60 after ischemia compared with the control group (Fig. 1).

Detection of Human Cells

Few HM immunoreactive cells (1.1 ± 0.9/mm²) were detected within the ipsilateral hemisphere of rats treated with hUTC. Double immunostaining revealed that HM-positive cells were not reactive for MAP2 or GFAP.

Effects of hUTC on SVZ Cell Proliferation

To examine whether administration of hUTC enhanced SVZ cell proliferation, BrdU-incorporating SVZ cells were measured at 60 days after stroke onset. Rats treated with hUTC exhibited a trend toward an increase in the density of BrdU-positive cells in the ipsilateral SVZ compared with the vehicle-treated rats (p = 0.07). However, the density of BrdU-positive cells in the contralateral SVZ was not significantly affected by the administration of hUTC (Fig. 2).

Effects of hUTC on Synaptophysin

Animals treated with hUTC exhibited significantly (p < 0.05) higher synaptophysin expression in the IBZ compared to rats treated with vehicle 60 days after stroke onset (Fig. 3).

Effects of hUTC on Vascular Density

Animals treated with hUTC showed significantly (p < 0.05) higher density of vWF-immunoreactive vessels in the IBZ compared with vehicle-treated rats (Fig. 4).

Discussion

Advanced age is associated with a decrease in brain plasticity and poor recovery from stroke (15). While previous studies have demonstrated beneficial effects of hUTC in experimental degenerative retinal and stroke models in young adult rodents (35,58), the efficacy of hUTC in aged rats with stroke was unknown. The present study demonstrates, for the first time, that intravenous administration of hUTC significantly improves neurological functional recovery in aged rats after stroke, which is accompanied by higher synaptophysin expression and vascular density in the IBZ. Hence, we conclude that the aged brain retains the capacity for neurorestoration in response to hUTC treatment.

Mesenchymal stem cells derived from human umbilical cord tissue have the ability to generate many cell types in vitro, including neurons and glia (19,39). These cells can transdifferentiate into neurons in experimental neurodegenerative diseases (31). However, Xiao et al. demonstrated that amelioration of neurological function following an intravenous injection of nonhematopoietic umbilical cord blood stem cells (nh-UCBSCs) into young rats after ischemic brain injury may be mediated by trophic actions that result in the reorganization of host nerve fiber connections within the injured brain (57). In the same study, it was observed that very few cells expressed the human nuclear marker within the brain, suggesting that the restorative effects of nh-UCBSCs may be mediated by mechanisms other than cell replacement. In the present study, only a very few hUTC were detected within the ischemic area. Double immunostaining revealed that these human cells did not coexpress neuronal antigens. In fact, it has been shown that subretinal infusion of hUTC preserves visual function in rats with retinal degeneration without neuronal differentiation, which is consistent with our current findings (35). Thus, our present data on the effectiveness of hUTC therapy for stroke obtained from older rats are consistent with the previous findings in young rats. Neither study presents evidence to support neuronal differentiation as the primary mechanism underlying the beneficial effects of hUTC (58). However, our results do favor the concept that these cells act as a paracrine mediator to elicit endogenous neurorestoration (10,13,32,40). Indeed, previous studies indicated that hUTC secrete several neurotrophic factors such as brain-derived neurotrophic factor (BDNF), fibroblast growth factor-2 (FGF-2), and interleukin-6 (IL-6), all of which have been shown to promote tissue repair after brain injury (32,35,47,55). Future studies are warranted to elucidate the dynamic distribution of hUTC, their secretion of paracrine factors, and their interactions with the host brain to foster neurorestoration.

We have previously shown higher synaptophysin expression and vascular density in young rats treated with hUTC after stroke (58). These processes may be key restorative mechanisms in response to hUTC treatment after the ischemic injury. However, age-associated impairments of synaptic plasticity and angiogenesis have been demonstrated by many experimental paradigms, which may lessen recovery from brain injury (2,26). Electrophysiological studies indicated that long-term potentiation/long-term depression (LTP/LTD), a cellular model of synaptic plasticity, is impaired during normal aging (8). In addition, although the aged brain is able to regain normal synaptic density after brain lesion, synaptogenesis is delayed in aged rats compared with young rats (2,26). Moreover, we and others have demonstrated that angiogenesis is impaired in aged rats after stroke (33,59). In the present study, animals treated with hUTC showed significantly higher synaptophysin expression and vessel density in the ischemic boundary area, which is associated with improved neurological functional recovery. Thus, although brain plasticity declines with age, our data suggest that the aged brain has the capacity for multiple regenerative processes in response to hUTC treatment, which likely contribute to functional recovery in the aged rat after stroke.

Intravenous administration of hUTC stimulates endogenous neural progenitor cell proliferation in young adult rats after stroke (58). However, in the present study, administration of hUTC resulted in a nonsignificant trend of increase in SVZ cell proliferation in the aged rats. The SVZ is the largest germinative layer of the adult brain (22), in which newly generated cells are capable of neuronal differentiation (1,17,45). Stroke triggers neurogenesis in the germinative regions in the young adult brain (4,28,38,59,60,62). However, stroke-induced neurogenesis is attenuated in the aged brain (11,15,27). Thus, the nonsignificant difference in SVZ cell proliferation in hUTC-treated aged animals is likely due to the natural attenuation of neurogenesis with increasing age. However, previous studies suggested that despite a reduction of cell proliferation in the SVZ of aged brain, the formation of mature striatal neurons after stroke appears similar in young and old rats (15), which argues against an intrinsic role of aging on neurogenesis after stroke. Thus, the effects of hUTC on endogenous neurogenesis and their relation to behavioral improvement in aged brain remain to be elucidated.

Neurological functional recovery after stroke is driven by neurorestorative processes including reorganization and rewiring of neuronal circuit, neurogenesis, and angiogenesis (5,13,14,41). We have previously demonstrated that intravenous administration of hUTC results in profound neurological functional improvement starting at 2 weeks after stroke onset, which is accompanied by enhancement of neurogenesis and synaptogenesis, as well as increased vessel density in the ischemic brain compared with the control (58). However, in aging rats, the behavioral benefits of hUTC's become evident in a delayed fashion. Advanced age may substantially influence the intrinsic remodeling capacity in response to ischemic insult (11,27), which may explain the delayed response to hUTC treatment as compared to young animals. Nevertheless, systemic hUTC treatment was still able to promote neurological functional improvement in aging rats after stroke.

In conclusion, for the first time, we demonstrate that intravenous administration of hUTC improves neurological function after stroke in a clinically relevant model of aged rats. We speculate that much of the beneficial effect of hUTC may be through the production of trophic factors, a mechanism that has been suggested to have the most immediate impact on stroke patients, rather than through cell replacement (12,13,24). As in the young animal treated with hUTC, in the aged animal, activation or enhancement of repair processes may be the underlying beneficial effect of hUTC in the treatment of stroke.

Footnotes

Acknowledgments

The research presented herein was funded by Advanced Technologies and Regenerative Medicine, LLC. The authors would like to acknowledge Yisheng Cui, M.D., Alex Zacharek, M.S., employees at Henry Ford Hospital, Anthony Kihm, Ph.D., Alex Harmon, Ph.D., David Colter, Ph.D., and Agnes Seyda, Ph.D., former employees at ATRM, for technical, editorial, and logistical support during the conduct of this study. K.H., B.K., M.R., and A.G. were employees of ATRM at the time the study was completed. (On December 30, 2012, ATRM merged into DePuy Orthopaedics, Inc.)

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