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Intracerebral transplantation of carotid body in rats with transient middle cerebral artery occlusion
Brain Research 1015 (2004) 50 – 56 www.elsevier.com/locate/brainres
Research report
Intracerebral transplantation of carotid body in rats with transient middle cerebral artery occlusion Guolong Yu a, Lin Xu a, Martin Hadman a, David C. Hess a,b, Cesar V. Borlongan a,b,c,* a
Department of Neurology, Medical College of Georgia, Augusta, GA 30912, USA Research and Affiliations Service Line, Augusta VAMC, Augusta, GA 30912, USA c Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912, USA b
Accepted 15 April 2004 Available online 1 June 2004
Abstract Recent laboratory and clinical studies demonstrate therapeutic efficacy of intracerebral transplantation of carotid body (CB) in Parkinson’s disease, possibly through secretion of neurotrophic factors. Here, we examined the role of CB in experimental stroke. In the first experiment, we hypothesized that removal of CB would exacerbate cerebral infarction and stroke-related behavioral deficits. Eight-week-old, male Sprague – Dawley rats were randomly divided into two groups: stroke with intact CB and stroke with surgically removed CB. We used the stroke model of temporary middle cerebral artery occlusion. The ipsilateral CB was removed in animals assigned to treatment group exposed to stroke with surgically removed CB. Behavioral tests, using the elevated body swing test, were conducted at days 1 – 3 after surgery. Cerebral infarction was visualized by TTC staining on day 3 post-surgery. The data revealed no significant differences in behavioral deficits and infarct volumes between the two groups. In the second experiment, CB cell suspension grafts or control adult tissue grafts were intracerebally transplanted into the ischemic penumbra immediately (within 1 h) after stroke surgery. The results revealed significant reduction of behavioral deficits and infarct volumes, accompanied by increased levels of neurotrophic factors, as detected by ELISA, in transplanted ischemic striatum collected from CB-grafted stroke animals. These observations suggest that surgical resection of CB in the periphery did not alter stroke pathology; however, CB when made available in the CNS, via intracerebral transplantation, could protect against stroke possibly through the synergistic release of neurotrophic factors. The present study extends the use of CB as efficacious graft source for transplantation. Published by Elsevier B.V. Theme: Development and regeneration Topic: Transplantation Keywords: Transplantation; Carotid body; Cerebral ischemia; Infarct volume; Middle cerebral artery occlusion; Neurotrophic factor
1. Introduction The decline in mortality from stroke has prompted efforts to develop effective rehabilitative therapies for stroke survivors. Despite considerable effort, no therapeutic approaches have been proven to be effective for the restoration of sensorimotor and cognitive deficits. Recent attention has focused on restoring brain function through cell transplantation [4,5,10,36,37]. Emerging animal studies * Corresponding author. Department of Neurology, BI-3080, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912-3200, USA. Tel.: +1-706-733-0188x2485; fax: +1-706-721-7619. E-mail address: [email protected] (C.V. Borlongan). 0006-8993/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.brainres.2004.04.055
have shown that cell transplantation leads to functional improvement in models of stroke, as well as in different models of neurological diseases [4,5,10,15,37,38]. In the laboratory, different types of graft sources such as fetal, adult striatal, cortical cells, genetically engineered cells and stem cells have been transplanted into experimental stroke animal models and have shown beneficial effects [4,5,10,15,37,38]. However, the difficulty in obtaining sufficient viable graft cells, as well as controversial ethical and legal issues limit the use of human fetal cells [6]. Finding efficacious alternative donor cell sources thus poses as a challenge for neural transplantation therapy. Neurotrophic factors play a critical role in neuronal development, as well as in protection and repair of the adult
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central nervous system. Recent laboratory and clinical studies have shown that intracerebral transplantation of genetically modified cells [18,30], fetal kidney tissue [7,20] or carotid body (CB) [1,25,34] ameliorates Parkinsonian deficits. A recent study reported by Espejo et al. [16] demonstrates therapeutic efficacy of intracerebral transplantation of CB in Parkinson’s disease (PD) possibly through secretion of neurotrophic factors [e.g., glial cell line-derived neurophophic factor (GDNF)], rather than the local release of dopamine. CB grafts have an extraordinary durability and can last almost the entire life of the animals. Moreover, a pilot study indicates that CB autograft transplantation is a relatively simple, safe, and viable treatment for PD patients [1]. No significant side effects were found after surgical resection of unilateral CB in rats and humans [1,16,21,25,32]. In addition, glomus cells of CB were verified to be particularly well suited for neural transplantation due to their extended viability in low oxygen condition [26,33,32]. Because of overlapping pathophysiologic mechanisms and precipitating factors between PD and stroke, such as increased oxidative stress and reduced levels of neurotrophic factors, cell transplantation proven effective in PD animal models and limited clinical trials, similarly may promote beneficial effects in stroke. Taken together, these studies on PD using neurotrophic factor therapy via cell transplantation suggest that intracerebral transplantation of CB may be a potential treatment strategy for stroke. Thus, we undertook two experiments: initially, we tested the hypothesis that removal of CB would exacerbate infarction and strokerelated behavioral deficits, and subsequently we assessed the feasibility and efficacy of CB as an alternative graft source in an animal model of transient middle cerebral artery occlusion. We also examined whether CB-mediated neurotrophic support is one of main mechanisms responsible for functional effects of intracerebral transplantation of CB.
2. Material and methods 2.1. Animals Eight-week-old, male Sprague – Dawley rats, weighing between 250 and 280 g, served as subjects for this study. Animals were kept under a 12:12-h light/dark cycle and allowed free access to food and water before and after surgical procedures. All experimental procedures followed the NIH guidelines for the use of animals in research to minimize discomfort in the animals during surgery and in the recovery period. All surgical procedures were conducted under aseptic conditions. 2.2. Experiment 1 2.2.1. Surgery Fourteen animals were randomly divided into two groups: stroke with intact CB and stroke with surgically removed CB
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(n = 7 for each group). We used the stroke model of temporary middle cerebral artery occlusion (MCAO) established by Koizumi et al. [24] and Nishino et al. [27] with minor modifications. Briefly, deeply anesthetized animals were placed in a supine position. After making an incision at the neck region, the junction between the internal and the external of the right carotid artery was exposed. The suture (17 mm non-absorbable nylon filament) was then introduced through the external carotid artery and guided into the internal carotid artery. This blocked the origin of the right MCA. During the 1-h MCA occlusion, the ipsilateral CB, firmly attached to the adventitia of the internal carotid artery near the bifurcation, was surgically removed in animals assigned to treatment group exposed to stroke with surgically removed CB. After the 1-h occlusion, the suture occluding the MCA was removed and the incision closed using routine procedures. Both MCA occlusion and surgical resection of CB was performed with no arterial bleeding. During all surgical procedures, care was taken to maintain the animals’ body temperature using a temperature-controlled heating pad. 2.2.2. Behavioral tests On days 1 – 3 after surgery, the animals were evaluated on the behavioral tests. Elevated body swing test (EBST) was performed to measure motor asymmetry as described previously [3]. It has been used to characterize a stable motor asymmetry in rats with successful occlusion of the MCA as early as 24 h and extending up to 6 months [2,19]. Briefly, the EBST involved handling the animal by its tail and recording the direction of the swings made by the animal. The test apparatus consisted of a clear Plexiglas box (40 40 35 cm). The animal was gently picked up at the base of the tail, and elevated by the tail until its nose was at a height of 2 in. (5 cm) above the surface. The direction of the swing, either left or right, was counted once the animal’s head moved sideways >10j from the midline of position of the body. After a single swing, the animal was placed back in the Plexiglas box and allowed to move freely for 30 s prior to retesting. These steps were repeated 20 times for each animal, while recording the direction of the frequency of each swing. 2.2.3. Measurement of infarct volume Infarction was visualized by staining brain slices with triphenyltetrazolium chloride (TTC), following a procedure described elsewhere [35,36]. Within 4 h after behavior testing on day 3 post-surgery, animals were anesthetized and then perfused intracardiacally with saline. The brain was then removed, immersed in cold saline for 10 min, and sliced in 2.0-mm-thick sections. The brain slices were incubated in 2% TTC dissolved in PBS for 20 min at 37 jC and then transferred to 4% paraformaldehyde solution for fixation. Infarction was revealed by a lack of TTC staining, which indicates that tissue is dehydrogenase deficient. The volume of infarction was measured in each slice and quantified by a computer-assisted image analysis system (NIH Image software, USA). The infarct volume
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was calculated by the following formula: 2 mm (thickness of the slice) [sum of the infarction area in all brain slices] [13]. 2.3. Experiment 2 2.3.1. Preparation and transplantation of CB cell suspension Carotid body tissues were harvested from ten 8-weekold, male Sprague –Dawley rats, weighing between 250 and 280 g. The procedure for CB resection was described in detail elsewhere [1]. Under aseptic conditions, CB was removed from animals and then placed on a Petri dish with aseptic Tyrode’s solution and isolated of surrounding fat and connective tissue. After isolation, the CB was mechanically dissociated and trypsinized to obtain cell suspension. Cells were then washed three times in sterile PBS, resuspended in 50 Al PBS, transferred to a 1.5-ml Eppendorf tube and kept on ice during transplantation. Each animal received approximately 200,000 viable cells in 3 Al PBS. Cell viability assay of cell aliquots, using Trypan blue exclusion method conducted immediately prior to and after transplantation surgery, revealed about 90% cell viability. Within 1 h after stroke surgery, randomly selected animals (n = 18 per group) received transplantation of CB cell suspension grafts or adult cerebellar cell suspension grafts (control adult tissue grafts) into the ischemic penumbra. The methods for preparation of control adult tissue have been reported in detail in our previous study [5]. Here, stroke animals received 200,000 viable cerebellar cells suspended in 3 Al PBS. We demonstrated previously that adult cerebellar grafts did not produce any observable deleterious or beneficial effects on stroke deficits [5]. All transplantation procedures were done in aseptic conditions. Individual animals were anesthetized and then mounted in a Kopf stereotaxic frame. The transplant material (CB cell suspension grafts or control adult tissue grafts in sterile saline) was then injected using a 25-gauge Hamilton syringe into the right striatum (AP= + 1.9 mm, ML = 1.5 mm, DV = 4.4 mm from bregma) based on the stereotaxic atlas by Paxinos and Watson [29]. This set of transplant coordinates targeted the medial striatum, which corresponded to the ischemic penumbra as revealed by our histologic findings from our previous studies [5,8,23]. Each animal received 3-Al injection over a 3-min period. The cannula of the Hamilton syringe was left in place for an additional 5min before retraction. During the transplant procedure, care was taken to maintain the animals’ body temperature using a temperature-controlled heating pad. 2.3.2. Behavioral tests and measurement of infarct volume On day 3 after surgery, the animals were evaluated on the behavioral tests. Within four hours after EBST, histological examination of brains from randomly selected animals (n = 9 per group) was performed using TTC staining. EBST and TTC assays were done as described in the first experiment.
2.3.3. Measurement of trophic factors by ELISA On day 3 after surgery, the remaining animals (n = 9 per group) were decapitated, their brains removed quickly and the striatum region containing the grafts was dissected and stored at 70 jC until analysis. Corresponding tissue samples from the contralateral intact striatum were also harvested and served as controls. Brains were homogenized using Teflon homogenizer (set 7, 20 strokes) in a lysis buffer [137 mM NaCl, 20 mM Tris, pH 8.0,1% NP-40, 10% glycerol, 1 mM phenylmentthyl-sulfonyl-fluoride (PMSF), 10 Ag/ml aptrotinin, 2 Ag/ml leupeptin,1 mM sodium vandate]. The homogenates were centrifuged at 12,000 g for 20 min. The pellets were discarded and the supernatant was acidified according to the method described by Okragly and Haak-Frendscho [28] with minor modification. The acidification was reported to enhance the detection of neurotrophic factors. Sample were then neutralized to pH 7.4 and then adjusted with buffer to contain the same amount of protein per ml (2 mg/ml). Protein concentrations were measured by using the BCA kit (Pierce, Rockford, IL). The samples were assayed for GDNF, NGF and BDNF by ELISA, using mouse monoclonal anti-GDNF, anti-NGF or anti-BDNF antibody (R&D Systems, USA) as a capture antibody and biotinylated goat anti-GDNF, anti-NGF or anti-BDNF antibody (R&D Systems, IL) as a detection antibody. A THERMOmax 96-well microplate reader (Molecular Devices, Sunnyvale, CA) was used to measure the optical densities. 2.4. Statistical analysis All data were expressed as mean F S.E.M. Statistical differences between treatment groups in EBST were analyzed using two-way ANOVA with post hoc Bonferroni ttest, while differences in GDNF, NGF and BDNF, and infarct volume were determined by Student’s t-test. A two-tailed p value of 0.05 or less was used to indicate statistical significance.
3. Result 3.1. Experiment 1: CB resection does not exacerbate strokeinduced motor deficits and cerebral infarction Across the 3-day post-surgery testing period, EBST results revealed motor deficits in both groups of stroke animals (Fig. 1, F2,21 = 1.36, p = 0.45). Stroke animals with surgically removed CB showed a pronounced biased swing activity that did not differ from those of the stroke animals with intact CB at any time point post-surgery ( p’s>0.05). On day 3 post-surgery, TTC staining in both groups of animals revealed a well-defined cerebral infarction predominantly within the striatum and overlying cortex ipsilateral to the occluded MCA (Fig. 2). Computer-assisted image analysis revealed that the cerebral infarction volume in stroke
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Fig. 1. CB resection does not exacerbate motor deficits. Stroke animals with surgically removed CB displayed pronounced motor asymmetry, as revealed by the elevated body swing test, over the 3-day post-surgery that did not significantly differ from stroke rats that had intact CB. Dashed line corresponds to swing activity ( F 8 S.E.M.) of normal non-ischemic animals from pilot studies.
animals that underwent CB resection was not significantly different from that of stroke animals with intact CB ( F1,12 = 0.41, p = 0.15). 3.2. Experiment 2 3.2.1. CB grafts reduce stroke-induced motor deficits and cerebral infarction Stroke animals that received intracerebral CB grafts displayed significant amelioration of motor asymmetry as revealed by EBST on day 3 post-surgery compared with stroke animals that received control adult tissue grafts (Fig. 3, F1,34 = 45.18, p < 0.001). In addition, CB grafts significantly decreased the cerebral infarct volume compared with control adult tissue grafts (Fig. 2, F1,16 = 27.44, p < 0.001).
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Fig. 3. CB cell grafts reduce motor deficits. Stroke animals that received CB cell grafts exhibited significantly less biased swing activity on day 3 postsurgery compared with stroke rats that received control grafts. *P < 0.05. Dashed line corresponds to swing activity ( F 8 S.E.M.) of normal nonischemic animals.
3.2.2. CB grafts elevate neurotrophic factors in CB-grafted stroke animals ELISA revealed significant treatment effects on protein levels of neurotrophic factors (Fig. 4, F 2,24 = 58.96, p < 0.001). Protein levels of GDNF, NGF, BDNF in the ipsilateral ischemic striatum collected from stroke animals that received CB grafts were significantly increased compared to the ipsilateral ischemic striatum stroke animals that received control adult tissue grafts ( p’s < 0.05). Varying degrees of protein elevation were noted as follows: 40%, 13% and 19% increments in GDNF, NGF and BDNF, respectively. Such elevation of neurotrophic factors in the ipsilateral ischemic striatal tissues collected from stroke animals that received CB grafts did not differ from contralateral intact striatum collected from stroke animals that received either CB grafts or control adult tissue grafts
Fig. 2. CB resection does not exacerbate cerebral infarction, but CB cell grafts reduce cerebral infarcts. Stroke animals with surgically removed CB exhibited marked cerebral infarction at day 3 post-surgery, as detected by TTC staining, that did not significantly differ from stroke rats that had intact CB. In contrast, stroke animals that received CB cell grafts showed significantly smaller cerebral infarcts on day 3 post-surgery compared with stroke rats that received control grafts. *P < 0.05.
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Fig. 4. CB cell grafts elevate neurotrophic factors. GDNF, NGF, and BDNF were significantly increased in the ischemic striata of stroke animals that received CB cell grafts compared with those from stroke animals that received control grafts. *P < 0.05. Dashed line corresponds to the arbitrary 100% ‘‘normal’’ level of trophic factors obtained from intact striatal tissues contralateral to the ischemic side of stroke animals.
( p’s>0.05). Post hoc correlational analysis, using Spearman rank correlation, revealed that such increased trophic factors inversely correlated with reduction in striatal infarct volume (Rho = 0.92, p = 0.02) in CB-grafted stroke animals.
4. Discussion The present study demonstrated that CB peripherally did not contribute to stroke pathology, but centrally CB cell grafts produced neuroprotection in acute stroke model. The observed attenuation of stroke-induced behavioral and histological alterations in stroke animals that received CB cell grafts was accompanied by increased levels of neurotrophic factors in the transplanted ischemic striatum. These data suggest that early intracerebral transplantation of CB cells protected against stroke possibly through the synergistic release of neurotrophic factors. Recent studies demonstrate that CB grafts can secrete a number of neurotrophic factors including GDNF, NGF, and BDNF [1,16,25,34]. Thus, we hypothesized that unilateral CB removal might exacerbate stroke-induced deficits as a consequence of reduced production of neurotrophic factors following surgery. Experiment 1 results, however, revealed that CB removal did not increase the volumes of cerebral infarction or worsen ischemia-related motor impairments. This observation parallels reports demonstrating no significant side effects following surgical resection of unilateral CB in human with carotid body tumor [21], as well as after unilateral CB removal for use in allotransplantation in animals [34], and for autotransplantation in PD patients [1]. We also observed (unpublished data) using ELISA analysis that levels of GDNF, NGF, BDNF in the striatum of stroke rats with CB removal did not significantly differ from that of stroke rats with intact CB. Thus, our results and those from other studies [21,34] indicate that surgical resection of CB does not produce central effects.
The immediate availability of neurotrophic factor-secreting CB cells centrally, however, is shown here to be beneficial in acute stroke. Experiment 2 results demonstrated that stroke rats that received CB cell grafts displayed significantly less asymmetry and reduced cerebral infarction compared to stroke animals that received control adult grafts. Moreover, ELISA revealed that the levels of GDNF, NGF, and BDNF in the CB transplanted ischemic striatum of stroke rats were significantly higher than those of stroke rats that received control adult cerebellar cell grafts. Such observation of elevated levels of neurotrophic factors in the CB transplanted stroke striatum accompanying behavioral and structural neuroprotection suggests that CB cell graftinduced functional effects are trophic factor-mediated events. The present neuroprotection produced by CB cell grafts supports accumulating evidence demonstrating similar beneficial effects of neural transplantation of other neurotrophic factor-secreting cells (e.g., fetal kidney tissue, fetal kidney cells and pineal gland graft) in stroke [8,10,13,23]. In addition, the neurotrophic factors shown here to be upregulated following CB transplantation have previously been demonstrated to exert neuroprotection via exogenous application [9,14,17]. For example, GDNF increased progenitor cells proliferation and differentiation in the lesioned striatum induced by MCA occlusion, thus maintaining a balance of Akt pathway and caspase cascade, and suppressing stroke-induced apoptotic cell death [22]. GDNF was also found to promote survival of transplanted cells by attenuating nitric oxide synthase activity and reducing reactive oxygen species formation [36]. Similarly, BDNF up-regulates the TrkB receptor of neurons residing along the ischemic penumbra thereby making them resistant to cell death produced by focal ischemia [31]. BDNF also enhances neuronal differentiation of transplanted cells in a stroke model [11], which could potentially enhance neuroprotection. Application of NGF also has been shown to enhance survival and neuronal differentiation of bone stromal marrow cells transplanted into the ischemic lesion [12]. A stable beneficial effect produced by a cell therapybased neuroprotection, such as the present approach, is likely dependent upon the continued release of neurotrophic factors from transplanted grafts. The present study provides a proof-of-principle evidence demonstrating that transplantation of CB cells into the ischemic striatum is effective in reducing infarct volume and behavioral abnormalities at least in the short-term. Additional laboratory studies are warranted to examine prolonged release of growth factors by CB cell grafts for long-term neuroprotection in stroke. Of note, long-term survival of CB grafts (up to 15 months) and their continuous secretion of trophic factors have been verified in a Parkinson’s disease model [12]. The present study is the first report of feasibility and efficacy of CB as an alternative graft source for neural transplantation in stroke. Experiments have demonstrated that exposure to one growth factor alone can support cell survival and growth [36,37]. However, the major limiting
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factor of neurotrophic factor treatment is the route of delivery; because the protein does not easily cross the blood – brain barrier, a direct infusion of growth factor is necessary. To achieve long-lasting protective effects will require multiple intracerebral infusions of the trophic factors. These logistical problems may hinder application of exogenous neurotrophic factor treatment in the clinic. It is likely that CB grafts may provide better neuroprotection than exogenously delivered growth factors. The production of a variety of growth factors is one of the advantages of CB cell grafts over multiple infusions of several trophic factors. The use of CB as autografts would be associated with less controversy and also may circumvent the need for immunosuppression. As shown in recent PD studies [1,16,25,32], we also demonstrated here that CB transplantation is a viable, relatively simple and safe procedure for stroke therapy. In summary, peripheral resection of CB did not alter stroke pathology; however, CB cells when made available in the CNS, via intracerebral transplantation, increased the levels of neurotrophic factors and protected against stroke. The present study extends the use of CB as efficacious graft source for transplantation therapy in stroke.
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Acknowledgements CVB is supported by VA VISN7 Career Development Award and American Heart Association Southeast Affiliate Grant-In-Aid Award.
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Research report
Intracerebral transplantation of carotid body in rats with transient middle cerebral artery occlusion Guolong Yu a, Lin Xu a, Martin Hadman a, David C. Hess a,b, Cesar V. Borlongan a,b,c,* a
Department of Neurology, Medical College of Georgia, Augusta, GA 30912, USA Research and Affiliations Service Line, Augusta VAMC, Augusta, GA 30912, USA c Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912, USA b
Accepted 15 April 2004 Available online 1 June 2004
Abstract Recent laboratory and clinical studies demonstrate therapeutic efficacy of intracerebral transplantation of carotid body (CB) in Parkinson’s disease, possibly through secretion of neurotrophic factors. Here, we examined the role of CB in experimental stroke. In the first experiment, we hypothesized that removal of CB would exacerbate cerebral infarction and stroke-related behavioral deficits. Eight-week-old, male Sprague – Dawley rats were randomly divided into two groups: stroke with intact CB and stroke with surgically removed CB. We used the stroke model of temporary middle cerebral artery occlusion. The ipsilateral CB was removed in animals assigned to treatment group exposed to stroke with surgically removed CB. Behavioral tests, using the elevated body swing test, were conducted at days 1 – 3 after surgery. Cerebral infarction was visualized by TTC staining on day 3 post-surgery. The data revealed no significant differences in behavioral deficits and infarct volumes between the two groups. In the second experiment, CB cell suspension grafts or control adult tissue grafts were intracerebally transplanted into the ischemic penumbra immediately (within 1 h) after stroke surgery. The results revealed significant reduction of behavioral deficits and infarct volumes, accompanied by increased levels of neurotrophic factors, as detected by ELISA, in transplanted ischemic striatum collected from CB-grafted stroke animals. These observations suggest that surgical resection of CB in the periphery did not alter stroke pathology; however, CB when made available in the CNS, via intracerebral transplantation, could protect against stroke possibly through the synergistic release of neurotrophic factors. The present study extends the use of CB as efficacious graft source for transplantation. Published by Elsevier B.V. Theme: Development and regeneration Topic: Transplantation Keywords: Transplantation; Carotid body; Cerebral ischemia; Infarct volume; Middle cerebral artery occlusion; Neurotrophic factor
1. Introduction The decline in mortality from stroke has prompted efforts to develop effective rehabilitative therapies for stroke survivors. Despite considerable effort, no therapeutic approaches have been proven to be effective for the restoration of sensorimotor and cognitive deficits. Recent attention has focused on restoring brain function through cell transplantation [4,5,10,36,37]. Emerging animal studies * Corresponding author. Department of Neurology, BI-3080, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912-3200, USA. Tel.: +1-706-733-0188x2485; fax: +1-706-721-7619. E-mail address: [email protected] (C.V. Borlongan). 0006-8993/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.brainres.2004.04.055
have shown that cell transplantation leads to functional improvement in models of stroke, as well as in different models of neurological diseases [4,5,10,15,37,38]. In the laboratory, different types of graft sources such as fetal, adult striatal, cortical cells, genetically engineered cells and stem cells have been transplanted into experimental stroke animal models and have shown beneficial effects [4,5,10,15,37,38]. However, the difficulty in obtaining sufficient viable graft cells, as well as controversial ethical and legal issues limit the use of human fetal cells [6]. Finding efficacious alternative donor cell sources thus poses as a challenge for neural transplantation therapy. Neurotrophic factors play a critical role in neuronal development, as well as in protection and repair of the adult
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central nervous system. Recent laboratory and clinical studies have shown that intracerebral transplantation of genetically modified cells [18,30], fetal kidney tissue [7,20] or carotid body (CB) [1,25,34] ameliorates Parkinsonian deficits. A recent study reported by Espejo et al. [16] demonstrates therapeutic efficacy of intracerebral transplantation of CB in Parkinson’s disease (PD) possibly through secretion of neurotrophic factors [e.g., glial cell line-derived neurophophic factor (GDNF)], rather than the local release of dopamine. CB grafts have an extraordinary durability and can last almost the entire life of the animals. Moreover, a pilot study indicates that CB autograft transplantation is a relatively simple, safe, and viable treatment for PD patients [1]. No significant side effects were found after surgical resection of unilateral CB in rats and humans [1,16,21,25,32]. In addition, glomus cells of CB were verified to be particularly well suited for neural transplantation due to their extended viability in low oxygen condition [26,33,32]. Because of overlapping pathophysiologic mechanisms and precipitating factors between PD and stroke, such as increased oxidative stress and reduced levels of neurotrophic factors, cell transplantation proven effective in PD animal models and limited clinical trials, similarly may promote beneficial effects in stroke. Taken together, these studies on PD using neurotrophic factor therapy via cell transplantation suggest that intracerebral transplantation of CB may be a potential treatment strategy for stroke. Thus, we undertook two experiments: initially, we tested the hypothesis that removal of CB would exacerbate infarction and strokerelated behavioral deficits, and subsequently we assessed the feasibility and efficacy of CB as an alternative graft source in an animal model of transient middle cerebral artery occlusion. We also examined whether CB-mediated neurotrophic support is one of main mechanisms responsible for functional effects of intracerebral transplantation of CB.
2. Material and methods 2.1. Animals Eight-week-old, male Sprague – Dawley rats, weighing between 250 and 280 g, served as subjects for this study. Animals were kept under a 12:12-h light/dark cycle and allowed free access to food and water before and after surgical procedures. All experimental procedures followed the NIH guidelines for the use of animals in research to minimize discomfort in the animals during surgery and in the recovery period. All surgical procedures were conducted under aseptic conditions. 2.2. Experiment 1 2.2.1. Surgery Fourteen animals were randomly divided into two groups: stroke with intact CB and stroke with surgically removed CB
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(n = 7 for each group). We used the stroke model of temporary middle cerebral artery occlusion (MCAO) established by Koizumi et al. [24] and Nishino et al. [27] with minor modifications. Briefly, deeply anesthetized animals were placed in a supine position. After making an incision at the neck region, the junction between the internal and the external of the right carotid artery was exposed. The suture (17 mm non-absorbable nylon filament) was then introduced through the external carotid artery and guided into the internal carotid artery. This blocked the origin of the right MCA. During the 1-h MCA occlusion, the ipsilateral CB, firmly attached to the adventitia of the internal carotid artery near the bifurcation, was surgically removed in animals assigned to treatment group exposed to stroke with surgically removed CB. After the 1-h occlusion, the suture occluding the MCA was removed and the incision closed using routine procedures. Both MCA occlusion and surgical resection of CB was performed with no arterial bleeding. During all surgical procedures, care was taken to maintain the animals’ body temperature using a temperature-controlled heating pad. 2.2.2. Behavioral tests On days 1 – 3 after surgery, the animals were evaluated on the behavioral tests. Elevated body swing test (EBST) was performed to measure motor asymmetry as described previously [3]. It has been used to characterize a stable motor asymmetry in rats with successful occlusion of the MCA as early as 24 h and extending up to 6 months [2,19]. Briefly, the EBST involved handling the animal by its tail and recording the direction of the swings made by the animal. The test apparatus consisted of a clear Plexiglas box (40 40 35 cm). The animal was gently picked up at the base of the tail, and elevated by the tail until its nose was at a height of 2 in. (5 cm) above the surface. The direction of the swing, either left or right, was counted once the animal’s head moved sideways >10j from the midline of position of the body. After a single swing, the animal was placed back in the Plexiglas box and allowed to move freely for 30 s prior to retesting. These steps were repeated 20 times for each animal, while recording the direction of the frequency of each swing. 2.2.3. Measurement of infarct volume Infarction was visualized by staining brain slices with triphenyltetrazolium chloride (TTC), following a procedure described elsewhere [35,36]. Within 4 h after behavior testing on day 3 post-surgery, animals were anesthetized and then perfused intracardiacally with saline. The brain was then removed, immersed in cold saline for 10 min, and sliced in 2.0-mm-thick sections. The brain slices were incubated in 2% TTC dissolved in PBS for 20 min at 37 jC and then transferred to 4% paraformaldehyde solution for fixation. Infarction was revealed by a lack of TTC staining, which indicates that tissue is dehydrogenase deficient. The volume of infarction was measured in each slice and quantified by a computer-assisted image analysis system (NIH Image software, USA). The infarct volume
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was calculated by the following formula: 2 mm (thickness of the slice) [sum of the infarction area in all brain slices] [13]. 2.3. Experiment 2 2.3.1. Preparation and transplantation of CB cell suspension Carotid body tissues were harvested from ten 8-weekold, male Sprague –Dawley rats, weighing between 250 and 280 g. The procedure for CB resection was described in detail elsewhere [1]. Under aseptic conditions, CB was removed from animals and then placed on a Petri dish with aseptic Tyrode’s solution and isolated of surrounding fat and connective tissue. After isolation, the CB was mechanically dissociated and trypsinized to obtain cell suspension. Cells were then washed three times in sterile PBS, resuspended in 50 Al PBS, transferred to a 1.5-ml Eppendorf tube and kept on ice during transplantation. Each animal received approximately 200,000 viable cells in 3 Al PBS. Cell viability assay of cell aliquots, using Trypan blue exclusion method conducted immediately prior to and after transplantation surgery, revealed about 90% cell viability. Within 1 h after stroke surgery, randomly selected animals (n = 18 per group) received transplantation of CB cell suspension grafts or adult cerebellar cell suspension grafts (control adult tissue grafts) into the ischemic penumbra. The methods for preparation of control adult tissue have been reported in detail in our previous study [5]. Here, stroke animals received 200,000 viable cerebellar cells suspended in 3 Al PBS. We demonstrated previously that adult cerebellar grafts did not produce any observable deleterious or beneficial effects on stroke deficits [5]. All transplantation procedures were done in aseptic conditions. Individual animals were anesthetized and then mounted in a Kopf stereotaxic frame. The transplant material (CB cell suspension grafts or control adult tissue grafts in sterile saline) was then injected using a 25-gauge Hamilton syringe into the right striatum (AP= + 1.9 mm, ML = 1.5 mm, DV = 4.4 mm from bregma) based on the stereotaxic atlas by Paxinos and Watson [29]. This set of transplant coordinates targeted the medial striatum, which corresponded to the ischemic penumbra as revealed by our histologic findings from our previous studies [5,8,23]. Each animal received 3-Al injection over a 3-min period. The cannula of the Hamilton syringe was left in place for an additional 5min before retraction. During the transplant procedure, care was taken to maintain the animals’ body temperature using a temperature-controlled heating pad. 2.3.2. Behavioral tests and measurement of infarct volume On day 3 after surgery, the animals were evaluated on the behavioral tests. Within four hours after EBST, histological examination of brains from randomly selected animals (n = 9 per group) was performed using TTC staining. EBST and TTC assays were done as described in the first experiment.
2.3.3. Measurement of trophic factors by ELISA On day 3 after surgery, the remaining animals (n = 9 per group) were decapitated, their brains removed quickly and the striatum region containing the grafts was dissected and stored at 70 jC until analysis. Corresponding tissue samples from the contralateral intact striatum were also harvested and served as controls. Brains were homogenized using Teflon homogenizer (set 7, 20 strokes) in a lysis buffer [137 mM NaCl, 20 mM Tris, pH 8.0,1% NP-40, 10% glycerol, 1 mM phenylmentthyl-sulfonyl-fluoride (PMSF), 10 Ag/ml aptrotinin, 2 Ag/ml leupeptin,1 mM sodium vandate]. The homogenates were centrifuged at 12,000 g for 20 min. The pellets were discarded and the supernatant was acidified according to the method described by Okragly and Haak-Frendscho [28] with minor modification. The acidification was reported to enhance the detection of neurotrophic factors. Sample were then neutralized to pH 7.4 and then adjusted with buffer to contain the same amount of protein per ml (2 mg/ml). Protein concentrations were measured by using the BCA kit (Pierce, Rockford, IL). The samples were assayed for GDNF, NGF and BDNF by ELISA, using mouse monoclonal anti-GDNF, anti-NGF or anti-BDNF antibody (R&D Systems, USA) as a capture antibody and biotinylated goat anti-GDNF, anti-NGF or anti-BDNF antibody (R&D Systems, IL) as a detection antibody. A THERMOmax 96-well microplate reader (Molecular Devices, Sunnyvale, CA) was used to measure the optical densities. 2.4. Statistical analysis All data were expressed as mean F S.E.M. Statistical differences between treatment groups in EBST were analyzed using two-way ANOVA with post hoc Bonferroni ttest, while differences in GDNF, NGF and BDNF, and infarct volume were determined by Student’s t-test. A two-tailed p value of 0.05 or less was used to indicate statistical significance.
3. Result 3.1. Experiment 1: CB resection does not exacerbate strokeinduced motor deficits and cerebral infarction Across the 3-day post-surgery testing period, EBST results revealed motor deficits in both groups of stroke animals (Fig. 1, F2,21 = 1.36, p = 0.45). Stroke animals with surgically removed CB showed a pronounced biased swing activity that did not differ from those of the stroke animals with intact CB at any time point post-surgery ( p’s>0.05). On day 3 post-surgery, TTC staining in both groups of animals revealed a well-defined cerebral infarction predominantly within the striatum and overlying cortex ipsilateral to the occluded MCA (Fig. 2). Computer-assisted image analysis revealed that the cerebral infarction volume in stroke
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Fig. 1. CB resection does not exacerbate motor deficits. Stroke animals with surgically removed CB displayed pronounced motor asymmetry, as revealed by the elevated body swing test, over the 3-day post-surgery that did not significantly differ from stroke rats that had intact CB. Dashed line corresponds to swing activity ( F 8 S.E.M.) of normal non-ischemic animals from pilot studies.
animals that underwent CB resection was not significantly different from that of stroke animals with intact CB ( F1,12 = 0.41, p = 0.15). 3.2. Experiment 2 3.2.1. CB grafts reduce stroke-induced motor deficits and cerebral infarction Stroke animals that received intracerebral CB grafts displayed significant amelioration of motor asymmetry as revealed by EBST on day 3 post-surgery compared with stroke animals that received control adult tissue grafts (Fig. 3, F1,34 = 45.18, p < 0.001). In addition, CB grafts significantly decreased the cerebral infarct volume compared with control adult tissue grafts (Fig. 2, F1,16 = 27.44, p < 0.001).
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Fig. 3. CB cell grafts reduce motor deficits. Stroke animals that received CB cell grafts exhibited significantly less biased swing activity on day 3 postsurgery compared with stroke rats that received control grafts. *P < 0.05. Dashed line corresponds to swing activity ( F 8 S.E.M.) of normal nonischemic animals.
3.2.2. CB grafts elevate neurotrophic factors in CB-grafted stroke animals ELISA revealed significant treatment effects on protein levels of neurotrophic factors (Fig. 4, F 2,24 = 58.96, p < 0.001). Protein levels of GDNF, NGF, BDNF in the ipsilateral ischemic striatum collected from stroke animals that received CB grafts were significantly increased compared to the ipsilateral ischemic striatum stroke animals that received control adult tissue grafts ( p’s < 0.05). Varying degrees of protein elevation were noted as follows: 40%, 13% and 19% increments in GDNF, NGF and BDNF, respectively. Such elevation of neurotrophic factors in the ipsilateral ischemic striatal tissues collected from stroke animals that received CB grafts did not differ from contralateral intact striatum collected from stroke animals that received either CB grafts or control adult tissue grafts
Fig. 2. CB resection does not exacerbate cerebral infarction, but CB cell grafts reduce cerebral infarcts. Stroke animals with surgically removed CB exhibited marked cerebral infarction at day 3 post-surgery, as detected by TTC staining, that did not significantly differ from stroke rats that had intact CB. In contrast, stroke animals that received CB cell grafts showed significantly smaller cerebral infarcts on day 3 post-surgery compared with stroke rats that received control grafts. *P < 0.05.
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Fig. 4. CB cell grafts elevate neurotrophic factors. GDNF, NGF, and BDNF were significantly increased in the ischemic striata of stroke animals that received CB cell grafts compared with those from stroke animals that received control grafts. *P < 0.05. Dashed line corresponds to the arbitrary 100% ‘‘normal’’ level of trophic factors obtained from intact striatal tissues contralateral to the ischemic side of stroke animals.
( p’s>0.05). Post hoc correlational analysis, using Spearman rank correlation, revealed that such increased trophic factors inversely correlated with reduction in striatal infarct volume (Rho = 0.92, p = 0.02) in CB-grafted stroke animals.
4. Discussion The present study demonstrated that CB peripherally did not contribute to stroke pathology, but centrally CB cell grafts produced neuroprotection in acute stroke model. The observed attenuation of stroke-induced behavioral and histological alterations in stroke animals that received CB cell grafts was accompanied by increased levels of neurotrophic factors in the transplanted ischemic striatum. These data suggest that early intracerebral transplantation of CB cells protected against stroke possibly through the synergistic release of neurotrophic factors. Recent studies demonstrate that CB grafts can secrete a number of neurotrophic factors including GDNF, NGF, and BDNF [1,16,25,34]. Thus, we hypothesized that unilateral CB removal might exacerbate stroke-induced deficits as a consequence of reduced production of neurotrophic factors following surgery. Experiment 1 results, however, revealed that CB removal did not increase the volumes of cerebral infarction or worsen ischemia-related motor impairments. This observation parallels reports demonstrating no significant side effects following surgical resection of unilateral CB in human with carotid body tumor [21], as well as after unilateral CB removal for use in allotransplantation in animals [34], and for autotransplantation in PD patients [1]. We also observed (unpublished data) using ELISA analysis that levels of GDNF, NGF, BDNF in the striatum of stroke rats with CB removal did not significantly differ from that of stroke rats with intact CB. Thus, our results and those from other studies [21,34] indicate that surgical resection of CB does not produce central effects.
The immediate availability of neurotrophic factor-secreting CB cells centrally, however, is shown here to be beneficial in acute stroke. Experiment 2 results demonstrated that stroke rats that received CB cell grafts displayed significantly less asymmetry and reduced cerebral infarction compared to stroke animals that received control adult grafts. Moreover, ELISA revealed that the levels of GDNF, NGF, and BDNF in the CB transplanted ischemic striatum of stroke rats were significantly higher than those of stroke rats that received control adult cerebellar cell grafts. Such observation of elevated levels of neurotrophic factors in the CB transplanted stroke striatum accompanying behavioral and structural neuroprotection suggests that CB cell graftinduced functional effects are trophic factor-mediated events. The present neuroprotection produced by CB cell grafts supports accumulating evidence demonstrating similar beneficial effects of neural transplantation of other neurotrophic factor-secreting cells (e.g., fetal kidney tissue, fetal kidney cells and pineal gland graft) in stroke [8,10,13,23]. In addition, the neurotrophic factors shown here to be upregulated following CB transplantation have previously been demonstrated to exert neuroprotection via exogenous application [9,14,17]. For example, GDNF increased progenitor cells proliferation and differentiation in the lesioned striatum induced by MCA occlusion, thus maintaining a balance of Akt pathway and caspase cascade, and suppressing stroke-induced apoptotic cell death [22]. GDNF was also found to promote survival of transplanted cells by attenuating nitric oxide synthase activity and reducing reactive oxygen species formation [36]. Similarly, BDNF up-regulates the TrkB receptor of neurons residing along the ischemic penumbra thereby making them resistant to cell death produced by focal ischemia [31]. BDNF also enhances neuronal differentiation of transplanted cells in a stroke model [11], which could potentially enhance neuroprotection. Application of NGF also has been shown to enhance survival and neuronal differentiation of bone stromal marrow cells transplanted into the ischemic lesion [12]. A stable beneficial effect produced by a cell therapybased neuroprotection, such as the present approach, is likely dependent upon the continued release of neurotrophic factors from transplanted grafts. The present study provides a proof-of-principle evidence demonstrating that transplantation of CB cells into the ischemic striatum is effective in reducing infarct volume and behavioral abnormalities at least in the short-term. Additional laboratory studies are warranted to examine prolonged release of growth factors by CB cell grafts for long-term neuroprotection in stroke. Of note, long-term survival of CB grafts (up to 15 months) and their continuous secretion of trophic factors have been verified in a Parkinson’s disease model [12]. The present study is the first report of feasibility and efficacy of CB as an alternative graft source for neural transplantation in stroke. Experiments have demonstrated that exposure to one growth factor alone can support cell survival and growth [36,37]. However, the major limiting
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factor of neurotrophic factor treatment is the route of delivery; because the protein does not easily cross the blood – brain barrier, a direct infusion of growth factor is necessary. To achieve long-lasting protective effects will require multiple intracerebral infusions of the trophic factors. These logistical problems may hinder application of exogenous neurotrophic factor treatment in the clinic. It is likely that CB grafts may provide better neuroprotection than exogenously delivered growth factors. The production of a variety of growth factors is one of the advantages of CB cell grafts over multiple infusions of several trophic factors. The use of CB as autografts would be associated with less controversy and also may circumvent the need for immunosuppression. As shown in recent PD studies [1,16,25,32], we also demonstrated here that CB transplantation is a viable, relatively simple and safe procedure for stroke therapy. In summary, peripheral resection of CB did not alter stroke pathology; however, CB cells when made available in the CNS, via intracerebral transplantation, increased the levels of neurotrophic factors and protected against stroke. The present study extends the use of CB as efficacious graft source for transplantation therapy in stroke.
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Acknowledgements CVB is supported by VA VISN7 Career Development Award and American Heart Association Southeast Affiliate Grant-In-Aid Award.
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