Long-term immunosuppression for CNS mouse xenotransplantation: Effects on nigrostriatal neurodegeneration and neuroprotective carotid body cell therapy

For Peer Review Long - term immunosuppre ssion for CNS mouse xenotransplantation: effects on nigrostriatal neurodegeneration and neuroprotective carotid body cell therapy. Journal: Xenotransplantation Manuscript ID XEN-18-O-0009.R1 Manuscript Type: Original Article Date Submitted by the Author: n/a Complete List of Authors: Villadiego, Javier; Instituto de Biomedicina de Sevilla, Cell Therapy and Molecular Physiology Lab; Universidad de Sevilla, Fisiología Médica y Biofísica Romo-Madero, Sonia; Instituto de Biomedicina de Sevilla; Universidad de Sevilla, Fisiología Médica y Biofísica García-Swinburn, Roberto; Instituto de Biomedicina de Sevilla; Universidad de Sevilla, Fisiología Médica y Biofísica Suárez-Luna, Nela; Instituto de Biomedicina de Sevilla; Universidad de Sevilla, Fisiología Médica y Biofísica Bermejo-Navas, Alfonso; Instituto de Biomedicina de Sevilla; Universidad de Sevilla, Fisiología Médica y Biofísica Echevarría, Miriam; Instituto de Biomedicina de Sevilla; Universidad de Sevilla, Fisiología Médica y Biofísica Toledo-Aral, Juan; Instituto de Biomedicina de Sevilla; Universidad de Sevilla, Fisiología Médica y Biofísica Keywords: Parkinson’s disease, Immunosuppression, Xenotransplantation, Carotid Body, Neurodegeneration. Topics: Organs - Other, Cells - Other (e.g. neural), Transplantation - small animal models, Immunosuppression Xenotransplantation Xenotransplantation For Peer Review 1 Long-term immunosuppression for CNS mouse xenotransplantation: effects on nigrostriatal neurodegeneration and neuroprotective carotid body cell therapy. Javier Villadiego 1,2,3 * ,# Sonia Romo-Madero 1,2, *, Roberto García-Swinburn 1,2 , Nela Suárez-Luna 1,2, , Alfonso Bermejo-Navas 1,2, , Miriam Echevarría 1,2, and Juan J. ToledoAral 1,2,3,# . 1. Instituto de Biomedicina de Sevilla-IBiS, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla. Sevilla, 41013. Spain. 2. Departamento de Fisiología Médica y Biofísica, Universidad de Sevilla. Sevilla, 41009. Spain. 3. Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain. *JV and SR-M contributed equally to this work. # Address correspondence to Dr. Juan J. Toledo-Aral or Dr. Javier Villadiego, Instituto de Biomedicina de Sevilla-IBiS, Avda. Manuel Siurot s/n, 41013 Sevilla, Spain. Tel: (34) 955923035; Fax: (34) 955923101; E-mail: [email protected] or [email protected] Running title: Long-term immunosuppression & antiparkinsonian xenografts. Page 1 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 2 Abstract Background: The use of long-term immunosuppressive treatments on neural transplantation has been controversial during the last decades. Although nowadays there is a consensus about the necessity of maintaining a permanent state of immunosuppression to preserve the survival of cerebral grafts, little is known about the effects that chronic immunosuppression produces both on the neurodegenerative process and on transplants function. Methods: Here we establish a new immunosuppressive protocol, based on the discontinuous administration of cyclosporine A (15 mg/Kg; s.c.) and prednisone (20 mg/Kg; s.c.), to produce long-term immunosuppression in mice. Using this treatment, we analyse the effects that long-term immunosuppression induces in a chronic 1methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) model of parkinsonism and on the neuroprotective and neurorestorative anti-parkinsonian actions exerted by rat carotid body (CB) xenografts. Results: This protocol preserves the survival of rat CB xenotransplants maintaining the general wellness of the grafted mice. Although permanent immunosuppression does not prevent the MPTP-induced cell death of nigral neurons and the consequent degeneration of dopaminergic striatal innervation, allowing for its use as Parkinson’s disease (PD) model, it reduces the microglial activation and slightly declines the striatal damage. Moreover, we reported that chronic administration of immunosuppressant drugs does not alter the neuroprotective and restorative anti-parkinsonian actions of rat CB xenografts into parkinsonian mice. Conclusions: This new immunosuppressive protocol provides a new murine model to assay the long-term effects of cerebral xenografts and offer a pharmacological alternative to the commonly used genetic immunodeficient mice, allowing the use of genetically modified mice as hosts. In addition it will permit the experimental analysis of the effects produced by human CB xenografts in the chronic PD murine model, with the final aim of using CB allografts as an option of cell therapy in PD patients. Page 2 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 3 Keywords: Parkinson’s disease, Immunosuppression, Xenotransplantation, Carotid Body, Neurodegeneration. Abbreviations: Carotid body, CB Cyclosporine A, CsA 3,4-dihydroxyphenylacetic acid, DOPAC Dopamine, DA Glial cell line-derived neurotrophic factor, GDNF Glial fibrillary acid protein, GFAP homovanillic acid, HVA 6-hydroxydopamine, 6-OHDA Ionized calcium-binding adapter molecule 1, Iba1 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine, MPTP Parkinson’s disease, PD Prednisone, Pred Substantia nigra pars compacta, SNpc Tyrosine hydroxylase, TH Page 3 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 4 1. Introduction Parkinson’s disease (PD) is characterized by the progressive degeneration of dopaminergic substantia nigra pars compacta (SNpc) neurons projecting to the striatum. This continuous loss of nigral neurons lead to a dopamine (DA) deficit in the striatum that correlates with the typical motor symptoms. 1-4 Although pharmacological and surgical therapies are currently used to palliate the symptoms, to date there is no cure for the disease. 5 During the last decades the replacement of the lost dopaminergic input by the transplantation of dopamine-releasing cells has been proposed as a treatment in PD patients. 6-8 Among the different cell types tested, the allograft of foetal ventral mesencephalic neurons provided the best clinical benefit. 9-13 However, the clinical efficacy of these grafts has been questioned by two double-blind trials that showed few clinical benefits with the appearance of dyskinesia in some of the grafted patients. 14,15 The variability in the clinical outcome obtained in the different trials has been attributed to different causes such as patient selection, tissue preparation or graft location. Nevertheless, the graft immunogenicity has emerged as a critical factor that could compromise the clinical benefit, since in the two double-blind trials that failed to show efficacy the patients were not immunosuppressed 14 , or immunosuppression was withdrawn after 6 months coinciding with the regression of the clinical benefit. 15 A different dopaminergic tissue used in antiparkinsonian cell therapy is the carotid body (CB), which is a bilateral organ located in the carotid bifurcation that contains a high number of dopaminergic cells. The intrastriatal graft of CB promotes a significant recovery in different preclinical models of PD, 16-20 which is mainly mediated by the release of the glial cell line-derived neurotrophic factor (GDNF) rather than the local release of dopamine by the transplant. 19-21 Two pilot clinical trials have also shown that CB autotransplantation can induce a clinical improvement in PD patients. However, the patient’s age and the progression status of the disease appear as important limitations for the clinical outcome. 22,23 For these reasons, although CB autotransplantation would appear as an attractive option because of the non-necessity to use Page 4 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 5 immunosuppression, the scarcity of the CB tissue obtained from the parkinsonian patient and putative alterations on the tissue integrity related with the aging and/or the parkinsonism would not recommend the CB autotransplantation, and clearly point to the need of developing allograft or xenografts of CB tissue. Moreover, a xenograft model in chronic parkinsonian mice will provide an excellent tool to increase the basic knowledge to improve future new clinical trials. Neuroinflammation and an altered immune response have been strongly linked to the progression of PD. 24-26 Cyclosporine A (CsA) has been demonstrated to produce some beneficial effects on 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6,- tetrahydropyridine (MPTP) of PD models. 27-29 . Indeed, either genetic or pharmacological manipulation of calcineurin, molecular target of CsA, induces certain levels of protection on α-synuclein based models of parkinsonism. 30-33 These findings suggest that the use of immunosuppressive treatments on allogenic or xenogenic cell based therapies for PD can affect the neurodegenerative model itself. However, in most of the preclinical studies that evaluate the effects of immunosuppression on PD models, the period of administration of immunosuppressive drugs is relatively short (ranged from 1 to 5 weeks), probably because of the difficulty to maintain the general wellness of experimental animals under severe pharmacological immunosuppression for long periods of time. Although some studies have analysed the long-term effects of immunosuppressive treatments on different rodent models, 34-36 it is necessary to study the potential alterations that continuous pharmacological immunosuppression can induce both in the chronic MPTP neurodegenerative process associated to PD and on the effects mediated by neurotrophic CB-based therapies. Here, we study the effects of long-term immunosuppression both on the neuropathological features of a new chronic MPTP mouse model based on the administration of low doses of MPTP for 3 months 37 and on the neuroprotective and reparative actions exerted by CB grafts on SNpc neurons. We used different immunosuppressive protocols and compare the general wellness of host mice, the Page 5 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 6 level of immunosuppression on the peripheral immune system and the preservation of rat CB xenograft, establishing a new immunosuppressed mouse model that allows to analyse the long-term effects of central nervous system xenografting. Moreover, we analysed the actions of long-term immunosuppression on the nigrostriatal degeneration and neuroinflammation induced by the chronic MPTP PD model. Finally, we demonstrated that rat CB xenografts, accompanied by chronic immunosuppression, showed similar neuroprotective and restorative effects on the nigrostriatal pathway of parkinsonian mice than the isogenic CB grafts previously reported by our group. 20 These findings clearly show that the beneficial actions induced by the CB xenografts are not affected by the chronic administration of immunosuppressive drugs. This offers a new experimental tool for the study of human CB xenografts in the chronic PD mice model, to better understand its mechanism of action and favour the possibility to use CB allografts as an option of cell therapy in PD patients. 2. Materials and Methods 2.1. Animal care and pharmacological treatments C57BL/6N male mice of 2-3 months of age (Charles River) were housed in a regulated temperature environment (22±1ºC) on a 12 h light/dark cycle, with ad libitum access to food and water. Mice were rendered parkinsonian, as previously described 20,37 by the subcutaneous (s.c.) administration of MPTP (20 mg/kg; Sigma) 3 times per week for 1, 2 or 3 months. The immunosuppression protocols applied were as follows: (i) “severe” immunosuppression, mice were subjected to daily administration of CsA (15 mg/Kg; s.c.) and Prednisone (Pred; 20 mg/Kg; s.c.); (ii) “moderate” immunosuppression, mice received daily injections of CsA (15 mg/Kg; s.c.) and Pred (20 mg/Kg; s.c.) during the first three weeks and alternant daily injections of CsA (15 mg/Kg; s.c.) or Pred (20 mg/Kg; s.c.) afterwards; and (iii) “mild” immunosuppression, animals received daily injections of CsA (15 mg/Kg; s.c.) and Pred (20 mg/Kg; s.c.) during the first two weeks, alternant daily injections of CsA (15 mg/Kg; s.c) or Pred (20 mg/Kg; s.c) for 1 week, Page 6 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 7 and 3 doses per week of CsA (15 mg/Kg; s.c ) afterwards. CsA and Pred were previously dissolved on NaCl 0.9% with 25% EtOH. In addition, as controls, mice were treated with the different combinations of vehicle solutions used in the MPTP and/or the immunosuppressive treatment: a group named “saline” received similar treatment as MPTP with saline solution (0.9% NaCl; Sigma); a “vehicle” group were only injected with the vehicle solution of the immunosuppressive treatment (0.9% NaCl, 25% EtOH; named as controls in the set of experiments exposed in Figures 1-2 and Supplementary Figures 1-2); a group named “saline+vehicle” was treated with both saline and vehicle solutions (named as controls in the set of experiments exposed in Figures 3-5). All control mice, treated with saline, vehicle, saline+vehicle, MPTP+vehicle and saline and “mild” immunosuppression were also analysed (Supplementary Figures 1 and 2). The total number of mice subjected to “mild” immunosuppression and/or MPTP treatment were as follows: controls (saline+vehicle), n=34; saline, n=12; vehicle, n=9; “mild” immunosuppression, n=15; MPTP, n=36; MPTP+vehicle, n=11; MPTP & Immsup, n=38. These mice were analysed with different experimental procedures on Figures 3-5, Supplementary Table 1 and Supplementary Figure 2. The general health status of the experimental mice were analysed by daily record of mortality, weight and, qualitatively, by the observation of any sign of dehydration or distress (assessed by the appearance of piloerection, coat staring, ocular and nasal discharges or aggressive behaviour. 38 At the end of the experiments, the animals were sacrificed under deep anaesthesia induced by a combination of 100 mg/kg ketamine (Pfizer) and 10 mg/kg xylazine (Bayer). All experiments were performed according to the European Directive 2010/63/EU and the Spanish RD/53/2013 for the protection of animals used for scientific purposes. The study was approved by the Animal Research Committee of the University Hospital Virgen del Rocío (University of Seville). Page 7 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 8 2.2. CB xenografting. All the mice grafted with rat CB xenografts received a CsA injection (15 mg/kg;s.c) 4-6 hours before initiating the surgical procedure. Intrastriatal CB grafting was performed as previously described. 19-21 In brief, rat carotid bifurcations were removed after neck incision. CBs were isolated and cleaned of surrounding tissue under a stereoscopic binocular microscope (Olympus SZX16). Then rat CBs were trimmed into 4-6 pieces and placed in 1 µl of Tyrode’s solution (140 mM NaCl; 4.7 mM KCl; 2 mM CaCl 2 ; 1 mM MgCl 2 ; 10 mM 4-2-hydroxyethyl-1-piperazineethanesulfonic acid, HEPES; 5 mM glucose, 5 mM pyruvate; Sigma). The xenografts were stereotaxically injected into the striatum (from bregma in mm: anteroposterior, +0.4; lateral, +2; ventral, -3.5) with a 25gauge syringe (Hamilton) according to the mouse brain stereotaxic atlas. 39 As an internal control, the contralateral striatum was injected with 1 µl of vehicle (named as sham side in the set of experiments exposed in Figure 6). The total number of CB xenografted animals and subjected to different pattern of immunosuppression and MPTP treatment were as follows: controls, n=25; severe immunosuppression, n=18; moderate immunosuppression, n=9; mild immunosuppression, n=20; MPTP + mild immunosuppression, n=21. These CB xenografted mice were analysed with the different experimental procedures on Figures 1,2,6 and Supplementary Figure 1. To avoid differences between animals, stereologic and densitometric values (see below) of CB xenografted parkinsonian mice are expressed as a percentage of the sham contralateral hemisphere. 2.3. Flow cytometry analysis. For the flow cytometry analysis 3 mice were measured for each time points and experimental conditions. For each mouse, 700 µl of blood were extracted from the left ventricle of immunosuppressed and control (treated with vehicle solution) mice with a heparinized syringe (heparin, 5000 U/ml; Applichem). The sample of blood was diluted with 700 µl of PBS (Sigma), mixed with 930 µl of Lymphocyte Separation Medium (LSM 1.077; Lonza) and centrifuged at 516 g for 25 minutes. The mononuclear Page 8 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 15 (Figure 4A-C) also showed a trend that suggest that long-term immunosuppressive treatment diminish the striatal DA depletion induced by the chronic MPTP treatment. In addition, the DOPAC/DA ratio, previously described as increased after chronic MPTP treatment and advanced PD, 37,47 is returned to the control levels after 3 months of immunosuppressive treatment (Fig. 4D). We also analysed the effect of the simple injection of the different control solutions used (saline, NaCl 0.9% for the MPTP treatment; and vehicle, EtOH 25% on NaCl 0.9% for the immunosuppressive treatment) both on controls and MPTP treated mice, not finding any significant consequences in the integrity of the dopaminergic nigrostriatal pathway. Moreover, chronic “mild” immunosuppression alone did not produce any significant alteration on the nigrostriatal pathway (measured by histological and neurochemical analysis; Supplementary Table 1 and Supplementary Fig. 2). The presence of clear signs of neuroinflammation, measured as an increase in reactive microglia and astrogliosis, is a well-established neuropathological feature of PD 25,48 that is clearly reproduced in our chronic MPTP parkinsonian model. 37 We investigated the effects of immunosuppression in the neuroinflammatory response induced by the chronic MPTP treatment at different time-points (1, 2 and 3 months). To evaluate the microglial activation, we labelled the microglial cells with the general marker Iba1 and performed size-dependent stereological quantification for the density of resting and active microglia. Although our “mild” immunosuppressive treatment did not produce any significant modification in the density of resting microglial cells either in the striatum or in the SNpc, it induced a reduction of the density of active microglial cells after MPTP treatment, which is clearly significant after 3 months of immunosuppression (Figure 5A-C). Regarding the characteristic astroglial response associated to MPTP induced nigrostriatal degeneration, the immunosuppressive-MPTP treated mice did not show any differences respect to the MPTP alone treated mice (Fig. 5D-E). Page 15 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 16 3.3. Dopaminotrophic actions of CB grafts under immunosuppressive treatment Striatal CB grafts from isogenic mice have been shown to produce both neuroprotection and restoration of chronic MPTP treated parkinsonian mice. 20 In addition, PD patients subjected to CB autograft showed some clinical benefits which are related with the age and the degree of parkinsonian affectation of the patient. 22,23 We analysed the effects of long-term immunosuppression (5 months) on the neuroprotective and restorative actions exerted by rat CB xenografts. That was performed to study if CB xenografts work ameliorating parkinsonism in the chronic MPTP murine model, in order to have an experimental tool to evaluate factors that could influence the clinical outcome of human CB transplants. To investigate the neuroprotective effect of CB xenografts we performed unilateral xenotransplants of rat CB, with a sham graft in the contralateral hemisphere as internal control, on receptor mice that were subjected to “mild” immunosuppression. Three weeks later, the CB xenografted mice were rendered parkinsonian by the chronic administration of MPTP (20 mg/Kg s.c.; 3 times/week, during 3 months), and were allowed to recover from the toxic treatment for 1 month. Afterwards, the CB xenografted mice were sacrificed and the histological examination of the nigrostriatal pathway was carried out (see experimental scheme in Fig. 6A). We found well-preserved intrastriatal CB xenografts in all the animals analysed (n=5), with abundant dopaminergic glomus cell. Moreover, the CB xenograft exerted a clear protection of ipsilateral TH + SNpc neurons that can be quantified with respect to the contralateral sham grafted hemisphere (145.6±5.2%; Fig. 6A,B). Consequently, the intrastriatal rat CB xenografts also induce a significant protection of the ipsilateral striatal dopaminergic innervation (Fig. 6A,B; 146.4±7.8% measured by TH + densitometry and 154.3±3.8% by stereological quantification of TH + fiber varicosities). To study if the striatal CB xenografts can also induce a restorative action (axonal sprouting) on the dopaminergic striatal innervation, we performed striatal CB Page 16 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 17 xenografts after the receptor mice were rendered parkinsonian by the chronic MPTP treatment. These xenografted CB mice were also immunosuppressed by the “mild” treatment after the transplantation (experimental scheme in Fig. 6C). All the mice analysed under these experimental design (n=6) also showed a well-maintained CB xenografts with numerous dopaminergic glomus cells. The striatal CB xenografts produced a significant increase in the dopaminergic striatal innervation in comparison with the contralateral sham grafted hemisphere (Fig 6C, D; 139.8±10.7% measured by TH + densitometry and 144.7±4.3% by stereological quantification of TH + fiber varicosities). This restorative effect produced by the CB xenografts on the striatal TH + fibers (after 3 months of chronic MPTP-treatment) occurs despite the fact that, as expected, the xenotransplants did not produce any beneficial effect on the number of TH + SNpc neurons (Fig. 6D, right panel). Thus, these results clearly indicate that in this experimental design the rat CB xenografts produce nigrostriatal fiber outgrowth because the CB xenografted striata have higher TH + innervation, respect to the sham grafted striata, with the same number of dopaminergic SNpc neurons. Interestingly, both neuroprotective and neurorestorative effect induced by the rat CB xenografts, under long-term immunosuppression, are qualitatively and quantitatively similar to the one previously reported by isogenic CB allotransplants in the same parkinsonian model and transplantations protocols, 20 indicating that the neurotrophic actions induced on the nigrostriatal pathway by CB cells are not affected by the use of chronic immunosuppression and xenografts. 4. Discussion The necessity of using long-term immunosuppression on neural transplantation strategies has been extensively discussed during the last decades. Although the brain has been considered an immune-privileged organ and some authors have reported neural graft survive in the absence of immunosuppressive treatment in monkeys and humans, 49-51 nowadays there is a broad consensus about that the lack of Page 17 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 18 immunosuppression produces an attenuation of the clinical outcome and possible adverse effects of neural transplants. 29,50,52,53 Different preclinical studies have analysed the effects of immunosuppression, based on CsA administration, on antiparkinsonian cell therapy. 29,46,54-59 However, in most of the studies using PD rodent models the period of CsA administration (1-6 weeks) is short, especially if it is compared with the slow and progressive natural course of the disease. This lack of studies that evaluate the long-term effects of immunosuppression on PD cell therapy are probably related with the adverse effects of drug treatments, 60,61 the mortality observed (>30%) and the high cost and difficulty to maintain the general wellness of animals subjected to severe pharmacological immunosuppression. In this work, we attempt to develop a new immunosuppressive treatment that combines the prevention of the immune rejection of the neural transplant with the long-term maintenance of the general wellness of the host animal. Among the different immunosuppressive treatments tested (severe, intermediate and mild) we show that the “mild” immunosuppression safeguards the viability of the CB xenografts for up to 5 months, with low mortality and minimal alterations of the wellness of host animals. This “mild” immunosuppressive pattern is based on the discontinuous administration of CsA, 3 injections of 15 mg/Kg per week, which reduces the dosage of the classical CsA administration pattern (ranged 15-25 mg/Kg per day) and presumably reduces the risk of kidney damage. 57 Moreover, this pattern of CsA administration produces a less accused reduction of the peripheral immunity but with a significant reduction of CD4 + T h lymphocytes which are considered the drivers of neural xenografts rejection. 62-64 This “mild” immunosuppression is sufficient to preserve the neural graft survival and functionality, preventing the appearance of infections or alterations on experimental animals. Interestingly, this pattern of “mild” immunosuppression could be used as a pharmacological alternative to the genetic immunodeficient mice to study the long-term effects of xenotransplantation, even in other body locations, allowing the use of genetically modified mice as hosts. Moreover, although our study has focused only in Page 18 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 19 the MPTP-mouse model, this “mild” immunosuppression protocol could be presumably also applied to other rat models of neurodegeneration. Different works have proposed that CsA treatment can produce some protection against 6-OHDA effects in rat and cellular PD models. 27,28,65 In addition, genetic or pharmacological inhibition of calcineurin induces protection on cellular and in vivo models of PD. 30-33 We analysed the effects of long-term “mild” immunosuppression on a chronic MPTP mouse model. Although we did not detect any protection of SNpc cell death, after 3 months of CsA treatment, there was a minor toxin-induced damage on dopaminergic striatal innervation. However, the fact that long-term immunosuppression diminished the dopaminergic striatal damage induced by chronic MPTP does not disallow this experimental model of parkinsonism, since it still presents a clear significant degeneration both in striatum and SNpc. These findings are in accord with the recent work of Tamburrino and colleagues where they postulate an improvement on axonal regeneration after 4 weeks of CsA treatment on the MPTP parkinsonian model. 29 Interestingly, the improvement on the striatal innervation after 3 months of CsA treatment is accompanied by a strong reduction on the microglial activation, suggesting that a decrease on the neuroinflammation associated to the neurodegenerative process could precede the improvement on the dopaminergic striatal innervation. Despite no PD patients have been treated with immunosuppressant drugs for enough time to unequivocally elucidate the impact on the disease course, PD patients subjected to sequential bilateral transplants, with immunosuppression, showed a bilateral motor recovery even before the second unilateral graft was carried out. 66 In conclusion, our data and other recent works in preclinical PD models 29,33 suggest that a “mild” and continuous immunosuppressive treatment can induce an improvement in PD patients through a decrease of the chronic neuroinflammation and an enhancement on the dopaminergic striatal function. Although this hypothesis is out of the scope of our study, it could be tested by appropriate clinical trials analysing the therapeutic effect of immunosuppressant drugs on PD patients. Page 19 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 20 Intrastriatal transplantation of CB cells have been shown to induce a significant histological and functional recovery in different preclinical models of parkinsonism. 1619,67-69 The mechanism underlying the anti-parkinsonian actions of CB transplants is a trophic support of the nigrostriatal pathway mediated, at least in part, by the release of GDNF. 19-21 Two pilot phase I/II open trials have shown that CB autotransplantation is a safe and feasible procedure that produce a clinical improvement in some of the patients, especially in the younger and less affected PD patients. 22,23 Here we studied the effects of the immunosuppressive treatment on the survival and anti-parkinsonian effects of rat CB xenografts, as a first study to analyse the CB xenografts potentiality in the chronic MPTP mouse model. Our results clearly showed the necessity of using immunosuppression to preserve the integrity of CB xenografts, suggesting the usage of immunosuppressant drugs in future trials that evaluate the clinical efficacy of CB allografts. A question that emerges from our study is the extraordinary high survival of CB tissue after intrastriatal transplantation, that has shown to be similar in rat, monkey and mice. 17-21 That contrasts with the recent study reported by Roberton and colleagues that clearly suggests that the mouse brain is an specially hostile environment for neural grafting. 35 This high survival of the CB after neural transplantation could be attributed to intrinsic properties of the CB tissue such as its physiological resistance to hypoxia 70 and oxidative stress 19 , and also to its high production of GDNF and other trophic factors 21,71 that could favour the graft survival by autocrine stimulation. In addition, we demonstrated that chronic CsA administration do not alter the neuroprotective and restorative actions that CB transplants exerts on the nigrostriatal pathway, showing the CB xenografts similar anti-parkinsonian effects than isogenic CB implants without immunosuppressive treatments. 20 The fact that “mild” immunosuppression preserves the integrity and the antiparkinsonian effects of the xenograft will permit, with future experiments, evaluate different factors that could modify the clinical efficacy of antiparkinsonian CB cell therapy performing human CB xenografts in the MPTP experimental model. In addition, these results open the Page 20 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 21 possibility of using human CB allografts, with permanent immunosuppression, as a realistic option to improve the clinical outcome of antiparkinsonian CB cell therapy. Acknowledgments The authors thank Dr. José López-Barneo for critical reading of the manuscript and general support. This study was supported by grants from the Spanish Government (PI12/02574-ISCIII; Red TerCel ISCIII RD12/0019/0033 and RD16/0011/0025; RTC2015-3309-1) and the Andalusian Government (Excellence project-P12-CTS-2739). Authorship JV, SRM and JJTA designed the experiments and interpreted the data. JV, SRM, RGS, NSL, ABN, ME and JJTA performed the experiments. JV and JJTA supervised all the experiments and wrote the manuscript. 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Cyclosporin A increases survival of cross-species intrastriatal grafts of embryonic dopamine-containing neurons. Exp Brain Res. 1985;60:204–208. Page 24 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 31 Supplementary Figure 1. Survival of rat CB xenografts and general wellness of host mice after 5 months of immunosuppression. A. Scheme of the experimental procedure carried out on mice CB xenografted and subjected to immunosuppression for 5 months. B. Image, after TH immunostaining, of an intrastriatal rat CB xenograft 5 months after transplantation, from a host mouse subjected to “mild” immunosuppression. Note the abundant presence of highly dopaminergic CB glomus cells. C,D. Kaplan-Meier (B) and weight (C) curves of mice grafted with rat CB xenotransplants treated with vehicle (control) or “mild” immunosuppression for 5 months. In the plots exposed in B and C, values of control mice are represented in purple and mice under “mild” immunosuppression in green. C: Data are presented as mean ± S.E.M. multiple unpaired two-tailed t-tests. B and C: control, n=5; mild, n=11. *p<0.05; **p<0.01; ***p<0.001. Supplementary Figure 2. Effects of chronic immunosuppression on the nigrostriatal pathway. A. Stereological quantification of TH + SNpc neurons of mice chronically treated with saline (controls) or saline and immunosuppression (Immsup) during 1, 2 and 3 months. B. Analysis of the striatal innervation by TH + striatal optical density (O.D.) measurements of the experimental groups exposed in A. C,D. Neurochemical analysis shown the striatal content of DA (C, left), DOPAC (C, central), HVA (C, right) and DOPAC/DA ratio (D) from animals treated during 3 months with the experimental conditions previously described. In the plots exposed in B and C, values of control mice are represented in purple and mice under immunosuppression in red. Data are presented as mean ± S.E.M. ANOVA test. A-D: A-D: 1 month: controls, n=3; Immsup, n=3. 2 months: controls, n=3; Immsup, n=4. 3 months: controls, n=3; Immsup, n=8. Page 31 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 32 Supplementary Table 1. Lack of effects of control solutions on nigrostriatal degeneration. A. Table showing the stereological quantification of TH + SNpc neurons of mice treated chronically with the control solutions of the MPTP (Saline; NaCl 0,9%) and/or immunosuppression (Vehicle; NaCl 0,9% + 25% EtOH) treatments. B. Table showing the striatal TH + optical density (O.D.) values (expressed as % of saline treated mice), after TH immunostaining, of the experimental groups previously described. Data are presented as mean ± S.E.M. ANOVA test with Bonferroni post hoc analysis. The number of animals analysed (n) are indicated in the tables. ***p<0.001 respect to the non-MPTP treated groups. Page 32 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 135x115mm (300 x 300 DPI) Page 33 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 81x147mm (300 x 300 DPI) Page 34 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 135x88mm (300 x 300 DPI) Page 35 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 77x83mm (300 x 300 DPI) Page 36 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 145x118mm (300 x 300 DPI) Page 37 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 141x151mm (300 x 300 DPI) Page 38 of 38 Xenotransplantation Xenotransplantation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60