Adrenal medulla of AS/AGU rats: a histological and immunohistochemical study.

BACKGROUND: The outcome of the autograft therapy for Parkinson's disease including autologous cells from adrenal medulla was disappointing. This could be attributed to the pathological process in Parkinson's disease affecting cells of the adrenal medulla. This study was performed to investigate the histopathological changes in the adrenal medulla of AS/AGU rat, a model of Parkinson's disease, in comparison with Albino Swiss (AS) rats. MATERIALS AND METHODS: A total of 24 male AS rats were divided into four groups, each of 6 animals: AS W1 - AS rats aged 1 week; AS adult - AS adult rats; AS/ /AGU W1 - AS/AGU rats aged 1 week; and AS/AGU adult - AS/AGU adult rats. The rats were sacrificed and the adrenal glands were dissected and processed for histological staining with haematoxylin and eosin and periodic acid Schiff and for immunohistochemical staining for S100 protein, ubiquitin and tyrosine hydroxylase. RESULTS: The histological investigation of the adrenal medulla of AS/AGU rats showed vascular congestion, inflammatory cellular infiltration, pyknotic nuclei, necrotic chromaffin cells and medullary inclusion bodies. The immunohistochemical investigation of AS/AGU rats showed a statistically significant decrease in the expression of S100 protein, ubiquitin and tyrosine hydroxylase compared to AS rats. CONCLUSIONS: The histological and immunohistological changes in the adrenal medulla could explain the failure of outcome of adrenal autograft therapy in Parkinson's disease.

Adrenomedullary progenitor cells: Isolation and characterization of a multi-potent progenitor cell population.

The adrenal is a highly plastic organ with the ability to adjust to physiological needs by adapting hormone production but also by generating and regenerating both adrenocortical and adrenomedullary tissue. It is now apparent that many adult tissues maintain stem and progenitor cells that contribute to their maintenance and adaptation. Research from the last years has proven the existence of stem and progenitor cells also in the adult adrenal medulla throughout life. These cells maintain some neural crest properties and have the potential to differentiate to the endocrine and neural lineages. In this article, we discuss the evidence for the existence of adrenomedullary multi potent progenitor cells, their isolation and characterization, their differentiation potential as well as their clinical potential in transplantation therapies but also in pathophysiology.

Past, present and future of human chromaffin cells: role in physiology and therapeutics.

Chromaffin cells are neuroendocrine cells mainly found in the medulla of the adrenal gland. Most existing knowledge of these cells has been the outcome of extensive research performed in animals, mainly in the cow, cat, mouse and rat. However, some insight into the physiology of this neuroendocrine cell in humans has been gained. This review summarizes the main findings reported in human chromaffin cells under physiological or disease conditions and discusses the clinical implications of these results.

Terapia celular para el tratamiento de la enfermedad de Parkinson / Cell therapy to treat Parkinson’s disease

En la enfermedad de Parkinson, las neuronas dopaminérgicas de la sustancia nigra degeneran, lo que trae como consecuencia un déficit de dopamina en sus áreas de proyección. Estas alteraciones histológicasy neuroquímicas se traducen en la mayoría de los trastornos motores que presentan los pacientes parkinsonianos. Las estrategias terapéuticasactuales se basan en fármacos que mejoran la neurotransmisión dopaminergica. Este enfoque terapéutico tiene efectos secundarios a largo plazo, como fl uctuaciones de la respuesta motora y discinesias. Eltrasplante de células dopaminérgicas fetales ha demostrado una mejoría de los síntomas clínicos. Actualmente, se desarrollan nuevas estrategiasterapéuticas para estimular un reemplazo neuronal endógeno a partir de precursores neuronales presentes en el cerebro adulto. Este trabajo resume los estudios que muestran el potencial de la terapia celular enla enfermedad de Parkinson

The pathological hallmark of Parkinson’s disease is a gradual loss of nigrostriatal dopamine-containing neurons, which is responsible for thecardinal motor symptoms of the disease. Current therapeutic strategies are mostly based on pharmacological enhancement of dopaminergicneurotransmission. This therapeutic approach has several long-term side effects, such as dyskinesias and fl uctuations of response, and is thereforelimited in its use. Transplantation of fetal dopaminergic precursor cells has provided the proof that a cell replacement therapy can ameliorate clinical symptoms in affected patients. Novel therapies aiming at a stimulation of an endogenous dopamine production within the brain at a continuous rate might provide a more physiological and elegant way to overcome the dopaminergic defi ciency in parkinsonian brains. Thisarticle will review recent studies demonstrating the potential of these alternative cell graft sources for treating Parkinson’s disease (AU)

Spinal subarachnoid adrenal medullary transplants reduce hind paw swelling and peripheral nerve transport following formalin injection in rats.

Previous studies have demonstrated that adrenal medullary chromaffin cells transplanted into the spinal subarachnoid space significantly reduced pain-related behavior following hind paw plantar formalin injection in rats. The data suggests a centrally mediated antinociceptive mechanism. The spinal transplants may have effects on sciatic nerve function as well. To address this, the current study examined the effects of spinal adrenal transplants on hind paw edema and the anterograde transport of substance P (SP) that occur following formalin injection. Robust formalin-evoked edema, as well as hind paw flinching, was observed in striated muscle control-transplanted rats, which were not observed in adrenal-transplanted rats. To visualize transport of SP, the sciatic nerve was ligated ipsilateral to formalin injection and the nerve was processed 48 h later for immunocytochemistry. A significant formalin-induced accumulation of SP immunoreactivity (IR) was observed proximal to the ligation in control-transplanted rats. In contrast, there was significantly less SP IR observed from nerve of adrenal-transplanted rats, suggesting a diminution of anterograde axoplasmic transport by adrenal transplants. The change in SP IR may have been due to an alteration of transport due to formalin injection, thus, transport was visualized by the accumulation of growth-associated protein 43 (GAP43) at the ligation site. Formalin injection did not significantly increase proximal accumulation of GAP43 IR, indicating that formalin does not increase anterograde transport. Surprisingly, however, adrenal transplants significantly diminished GAP43 IR accumulation compared to control-transplanted rats. These data demonstrate that spinal adrenal transplants can attenuate the formalin-evoked response by modulating primary afferent responses.

Neuropathological study 16 years after autologous adrenal medullary transplantation in a Parkinson's disease patient.

To date, there is no clinicopathological correlation of adrenal medullary transplant cases in patients with survival beyond a few years. Postmortem examination of a brain from a patient with Parkinson's disease (PD), 16 years after autologous adrenal medullary transplant, was performed using tyrosine hydroxylase (TH) and chromogranin A. The patient experienced a four-year initial improvement in motor function followed by resumption of the progressive nature of her disease that continued until her death. She expired 16 years following grafting. At autopsy, TH stain of the brain revealed severe loss of TH-immunoreactivity in the substantia nigra and Lewy bodies, confirming the diagnosis of PD. The transplant site was identified by the presence of scarring and there was complete absence of any TH staining cells at the site of the transplant. There were few surviving cells staining with chromogranin A. The absence of TH-staining cells in the transplant 16 years after surgery provides further evidence that adrenal medullary transplants do not survive in the long term.

[Cell therapy and other neuroregenerative strategies in Parkinson's disease (I)]. / Terapia celular y otras estrategias neurorregenerativas en la enfermedad de Parkinson (I).

OBJECTIVE: To review, from a mainly clinical standpoint, the different strategies applied to regenerate or restore the nigrostriatal dopaminergic system in Parkinson's disease. This first part focuses on the results of adrenal medulla and human fetal mesencephalic transplants, and a second part will address transplants of other cell types, administration of trophic factors, and gene therapy. DEVELOPMENT: Adrenal medulla transplants were abandoned because of their inconsistent results and high morbidity. Although fetal mesencephalic transplantation can produce long-term restoration of striatal dopamine deficiency, this neurochemical effect is clinically inadequate in presence of progressive neuronal loss. Other strategies with similar 'dopaminergic' action mechanism are not a therapeutic option in this setting. CONCLUSIONS: The objective of neuroregenerative therapy for Parkinson's disease should include trophic restoration of damaged neuronal systems, since improvement in striatal dopaminergic function is not sufficient. After the recent failure of the direct (intraventricular or intraputaminal) administration of glial cell line-derived neurotrophic factor (GDNF), attention of researchers has focused on indirect methods, including transplantation of GDNF-producing cells (carotid body cell aggregates or different genetically modified cells, including stem cells), and in vivo gene therapy.

Terapia celular y otras estrategias neurorregenerativas en la enfermedad de Parkinson (I) / Cell therapy and other neuroregenerative strategies in Parkinson’s disease (I)

Objetivo. Revisar, desde una perspectiva fundamentalmente clínica, las diferentes estrategias que tienen como finalidad la regeneración o restauración del sistema dopaminérgico nigroestriatal en la enfermedad de Parkinson. En esta primera parte se analizan los resultados de los trasplantes de médula adrenal y de mesencéfalo fetal humano, y se continúa en una segunda parte con los trasplantes de otras estirpes celulares, la administración de factores tróficos y la terapia génica. Desarrollo. Los trasplantes de médula adrenal se abandonaron por la inconsistencia de sus resultados y su elevada morbilidad. Los trasplantes de mesencéfalo fetal poseen capacidad para restaurar a largo plazo el déficit de dopamina estriatal; sin embargo, este efecto neuroquímico ha demostrado ser clínicamente insuficiente en presencia de una degeneración neuronal progresiva. En este contexto, otras estrategias con similar mecanismo de acción ‘dopaminérgico’ no suponen una alternativa. Conclusiones. El objetivo de una auténtica terapia neurorregenerativa para la enfermedad de Parkinson debería ser no sólo incrementar la función dopaminérgica estriatal, sino promover la restauración trófica de los sistemas neuronales dañados. Tras el reciente fracaso de los métodos de administración directa (intraventricular e intraputaminal) de factor neurotrófico derivado de una línea celular glial (GDNF), ha cobrado una mayor relevancia la investigación de otros métodos indirectos, como el trasplante de células productoras de GDNF (agregados celulares del cuerpo carotídeo, distintas células modificadas genéticamente) y la terapia génica in vivo

Objective. To review, from a mainly clinical standpoint, the different strategies applied to regenerate or restore the nigrostriatal dopaminergic system in Parkinson’s disease. This first part focuses on the results of adrenal medulla and human fetal mesencephalic transplants, and a second part will address transplants of other cell types, administration of trophic factors, and gene therapy. Development. Adrenal medulla transplants were abandoned because of their inconsistent results and high morbidity. Although fetal mesencephalic transplantation can produce long-term restoration of striatal dopamine deficiency, this neurochemical effect is clinically inadequate in presence of progressive neuronal loss. Other strategies with similar ‘dopaminergic’ action mechanism are not a therapeutic option in this setting. Conclusions. The objective of neuroregenerative therapy for Parkinson’s disease should include trophic restoration of damaged neuronal systems, since improvement in striatal dopaminergic function is not sufficient. After the recent failure of the direct (intraventricular or intraputaminal) administration of glial cell line-derived neurotrophic factor (GDNF), attention of researchers has focused on indirect methods, including transplantation of GDNF-producing cells (carotid body cell aggregates or different genetically modified cells, including stem cells), and in vivo gene therapy

Inhibitory effects of adrenomedullary hormone on the induction and growth of fibrosarcoma by methylcholanthrene.

OBJECTIVE: Effects of adrenomedullary hormone(s) on the induction and growth of fibrosarcoma by methylcholanthrene (MC) were examined. METHODS: At 28 days of age, male Wistar rats were divided into four groups: 1) control, 2) bilateral adrenomedullectomy (Bil. AMX), 3) right AMX + left adrenomedullary autotransplantation (AMX + AMT), 4) Bil. AMX + epinephrine injection (Bil. AMX + E) groups. 14 days after surgery, MC crystals were inserted underneath the dorsal skin, and in the Bil. AMX + E group, epinephrine was injected subcutaneously, twice every week. RESULTS: The incidence of tumor at 90 days after the MC injection was 8 per 35 cases (22.9%) in the control group, 12 per 36 cases (33.3%) in the AMX + AMT group, 8 per 28 cases (28.6%) in the Bil. AMX + E group, and each value was lower compared with that of the Bil. AMX group, 24 per 34 cases (70.6 %), (P<0.001, P<0.002, P<0.005). Such differences among groups were not seen at 165 days after the injection of MC. DISCUSSION: The mechanisms of effects of AMX, AMT and/or epinephrine on the tumor incidence have been discussed with reference to tumor promotion, vascular neoplasia, etc. Since norepinephrine remaining in the blood of AMX rats was ineffective, at least it is likely that this inhibitory effect of epinephrine is mediated via the beta2-receptor. CONCLUSION: The results suggest that adrenomedullary hormone, probably epinephrine, has inhibitory effects on the induction and growth of fibrosarcoma by MC, particularly in the early stage.

Enhanced antinociception by nicotinic receptor agonist epibatidine and adrenal medullary transplants in the spinal subarachnoid space.

Adrenal medullary transplants in the spinal subarachnoid space can reduce nociception, via the release of catecholamines and other analgesic substances, and this may be enhanced by stimulation of transplanted chromaffin cell surface nicotinic acetylcholine receptors (nAChRs). In addition, spinal nAChRs have been implicated in modulating nociception and can interact synergistically with alpha-adrenergic agents. Thus, enhanced antinociception by potent nAChR agonists such as frog skin derivative epibatidine in adrenal-transplanted animals could potentially occur via multiple mechanisms, including nicotinic-alpha-adrenergic synergy and stimulation of chromaffin cell nicotinic receptors. In order to test this, male Sprague-Dawley rats were implanted with intrathecal catheters and either adrenal medullary or control striated muscle transplants in the spinal subarachnoid space at the lumbar enlargement. Animals were tested for nociceptive responses before and after intrathecal injection of several doses of epibatidine using acute analgesiometric tests (tail flick, paw pressure) and the formalin test. After adrenal medullary, but not control, transplantation, nociceptive thresholds to acute noxious stimuli were slightly but consistently elevated, and phase 2 formalin responses decreased. Following intrathecal injection of epibatidine, acute nociceptive response latencies were modestly elevated and phase 2 formalin flinches modestly suppressed in control animals, but only at the highest dose test, with some attendant motor side-effects. In contrast, in adrenal medullary-transplanted animals, epibatidine elevated responses to acute noxious stimuli and markedly suppressed phase 2 formalin responses in a dose-related fashion. The enhanced antinociceptive effect following epibatidine was attenuated with either nAChR antagonist mecamylamine or alpha-adrenergic receptor antagonist phentolamine. The current results demonstrate that intrathecal injection of the nAChR ligand epibatidine can produce significant antinociception in adrenal-transplanted rats in both acute and tonic nociceptive tests and suggest that the use of nicotinic agents in combination with adrenal medullary transplantation could provide maximal therapeutic benefit by synergistically improving antinociception while avoiding the detrimental side-effects of these agents.

Effects of adrenal medulla graft on recovery of GABAergic and dopaminergic neuron deficits in mice: behavioural, pharmacological and immunohistochemical study.

We studied the capacity of adrenal medullary transplant to restore the deficits of GABAergic and dopaminergic neurons in mice injected with quinolinic acid (QA), using an open field test as well as pharmacological and immunohistochemical techniques. We analysed behavioural traits-total locomotor activity, peripheral and central activities, grooming, leaning and rearing in the QA-lesioned mice and mice that had undergone adrenal medulla (AM) transplantation. We found that the adrenal transplant recovered a loss of GABAergic neurons. It reduced QA-induced hyperactivity in locomotion and improved emotional indices. In addition, immunohistochemical studies of catecholaminergic markers-tyrosine hydroxylase (TH), dopamine (DA) and neuronal vesicular monoamine transporter type 2- and a single post-trial injection of tetrabenazine (TBZ; 5 mg/kg) indicated that catecholamines-synthesising chromaffin cells in the AM grafts were also involved in the beneficial effects. A likely interpretation of this behavioural pattern of results is that adrenal medullary transplants set into play an interaction between GABAergic and DAergic factors. Our results may contribute to the clarification of the beneficial effects of AM transplants in striatal function.

Cellular therapies for spinal cord injury: what will the FDA need to approve moving from the laboratory to the human?

The transplantation of living cells and tissues to restore function and/or provide therapeutic molecules has been an active and ongoing area of research interest for over 25 years. Several of these potential therapies have reached initial clinical trials, and it is likely that applications will continue to expand, and that novel and improved approaches will be explored over the next several years. In the past, many of these experimental approaches were tested in early clinical trials without the oversight of regulatory agencies such as the Food and Drug Administration. However, as novel cellular therapies move from preclinical laboratory findings to the clinical arena, researchers and regulators face new and continually evolving issues and uncertainties involving long-term safety and efficacy. Using adrenal medullary transplantation in the spinal cord for pain as an example, this review presents an overview of past and current regulatory guidelines for moving these promising, novel cellular transplantation therapies from the laboratory to the human.

Clinical neurotransplantation: core assessment protocol rather than sham surgery as control.

Basic neurotransplantation research evoked clinical trials of restorative brain surgery. Parkinson's disease was the first and primary test bed for this putative new therapeutic method. Various centers performed the grafting surgery and the behavioral evaluations in different ways, and observed a varying degree of symptomatic relief. This led to a plea for double blind placebo-controlled clinical trials, which have since been performed and of which the first outcomes were recently published. In the present paper this approach of experimental neurotransplantation in brain diseases is discussed and rejected. Neural grafting in the central nervous system is irreversible and is therefore not suitable for experimental approaches originally designed for and best suited to drug studies. For Parkinson's disease in particular, the technique is far from optimized to perform large-scale studies at this stage. Moreover, previous negative results of adrenal medulla tissue implantation in the brain of patients make placebo effects rather unlikely. Moral arguments concerning the validity of the informed consent, therapeutic misconception, and the risk/benefit ratio can be added in the plea against this control surgery. Finally, a recommendation is made for study designs that apply a disease-dedicated core assessment protocol (CAP) that can evaluate the period from pre-operative to post-convalescent stages quantitatively, and therefore, unbiased. The strength of these CAPs is that they allow comparisons of different grafting techniques, of results between centers and of other types of interventions and invasive treatments such as deep brain stimulation. On ethical grounds, it is unacceptable not to use a study design that circumvents sham or imitation surgery. It is a challenge for the neuroscience community to develop CAPs for brain diseases that are eligible for neurotransplantation in the future.

Adrenal medullary transplants reduce formalin-evoked c-fos expression in the rat spinal cord.

Previous studies have indicated that adrenal medullary chromaffin cells transplanted into the spinal subarachnoid space can alleviate pain behaviors in several animal models. The goal of this study was to assess whether decreased activation of spinal dorsal horn neurons responsive to nociceptive stimuli may contribute to these antinociceptive effects. In order to address this, expression of neural activity marker c-fos in response to intraplantar formalin was evaluated in animals with intrathecal adrenal medullary or control striated muscle transplants. Adrenal medullary transplants significantly attenuated formalin-induced flinching behaviors in both acute and tonic phases of the formalin response, in comparison with control transplanted animals. Fos-like-immunoreactive (Fos-LI) cell numbers were markedly reduced in the dorsal horns of animals with adrenal medullary transplants in comparison to robust Fos-LI expression in control transplanted animals. This reduction was observed in both superficial and deep laminae of the dorsal horn, but the magnitude of the decrease was greatest in lamina V. Similar to reports using other antinociceptive treatments, some residual c-fos expression was observed, particularly in laminae I-II, in animals with adrenal medullary transplants. The results of these studies suggest that adrenal medullary transplants produce antinociception in part by inhibiting spinal dorsal horn neuronal activation in response to noxious stimuli.

Functional regeneration in a rat Parkinson's model after intrastriatal grafts of glial cell line-derived neurotrophic factor and transforming growth factor beta1-expressing extra-adrenal chromaffin cells of the Zuckerkandl's organ.

Intrabrain transplantation of chromaffin cell aggregates of the Zuckerkandl's organ, an extra-adrenal paraganglion that has never been tested for antiparkinsonian treatment, induced gradual improvement of functional deficits in parkinsonian rats. These beneficial effects were related to long survival of grafted cells, striatal reinnervation, and enhancement of dopamine levels in grafted striatum. Grafted cells were not dopaminergics, but they expressed glial cell line-derived neurotrophic factor (GDNF) and transforming growth factor-beta(1). These factors were detected in the host striatal tissue, indicating that chromaffin cells secreted them after grafting. Because glial cell line-derived neurotrophic factor possesses neurorestorative properties over dopaminergic neurons, and transforming growth factor-beta(1) is a cofactor that potentiates the neurotrophic actions of GDNF, functional regeneration was likely caused by the chronic trophic action of neurotrophic factors delivered by long-surviving grafted cells. This work should stimulate research on the clinical applicability of transplants of the Zuckerkandl's organ in Parkinson's disease.

Spinal allografts of adrenal medulla block nociceptive facilitation in the dorsal horn.

Transplantation of chromaffin cells into the lumbar subarachnoid space has been found to produce analgesia, most conspicuously against chronic neuropathic pain. To ascertain the neurophysiological mechanism, we recorded electrical activity from wide-dynamic-range dorsal horn neurons in vivo, measuring the short-lasting homosynaptic facilitatory effect known as windup, which is induced by repetitive C-fiber input. Rats were given adrenal medulla allografts, or, as controls, striated-muscle allografts. The adrenal-transplanted rats showed analgesia 3--4 wk after transplantation, measured as a reduction in flinching reflexes 30--55 min after subcutaneous formalin injection. Recordings were made under halothane anesthesia, 3--7 days following the behavioral testing. The average C-fiber response and subsequent afterdischarge were facilitated severalfold in control rats by 1-Hz cutaneous electrical stimulation. Such facilitation was essentially absent in adrenal-transplanted animals and also in the A-fiber response of both preparations. Extirpation of transplanted tissue several hours prior to recording did not significantly affect this difference. In conclusion, the adrenal transplants block short-term spinal nociceptive facilitation, probably by stimulating some persistent cellular process that may be an important determinant, but not the only one, of their analgesic effect.

Involvement of adrenal medulla grafts in the open field behavior.

Immunohistochemical and behavioral techniques were used to study the effects of adrenal medulla grafts, implanted in striatum after bilateral kainic acid (KA) lesions of this structure, on the open field behavior of mice. KA-induced behavioral changes in leaning, grooming and locomotor activity of the open field test were significantly improved after grafting of the adrenal medulla, and in some respects, fully restored. Immunohistochemical identification showed that grafts contained neuron-like cells with a tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase, gamma-aminobutyric acid (GABA), choline acetyltransferase (ChAT), and enkephalin-like immunostainings. A likely interpretation of this complex pattern of results is that adrenal medullary grafts may restore the deficits of GABAergic neurons which in turn reverse the abnormalities in emotionality and locomotion. Neurobiologically, these behavioral improvements probably involve GABAergic and catecholaminergic factors of adrenal medulla grafts, although other neuroactive substances, such as acetylcholine and enkephalins, cannot be excluded.