Showing papers in "Neural Plasticity in 1992"
TL;DR: A method of examining behavior in unilateral 6-OHDA rats which addresses concerns of severe and chronic akinesia and a reduction of the contraversive circling induced by the DA receptor agonist apomorphine or of the ipsiversive circlinginduced by amphetamine is described.
Abstract: In transplantation research on Parkinson’s disease, behavioral evidence of graft-specific improved function is as necessary as anatomical or chemical data in evaluating the success of the procedure. The circling model of Ungerstedt has been widely adopted to provide such evidence, in part because it is so convenient and (relative to primate models) inexpensive. In this model 6hydroxydopamine (6-OHDA) is used to deplete nigrostriatal dopamine (DA) unilaterally. A reduction of the contraversive circling induced by the DA receptor agonist apomorphine or of the ipsiversive circling induced by amphetamine has been useful as a screen to test the integrity of grafts. However, it would be more compelling if the model included akinesia, one of the most disruptive symptoms of the disease. In addition, the model should reflect the clinical finding that akinesia becomes increasingly resistant to DAergic agonists, particularly in the severe later stages of parkinsonism. In the unilateral 6OHDA rat, DA-agonist induced circling is actually more vigorous when the depletion of DA is more severe. Moreover, during circling behavior all limbs make stepping movements, even the limbs contralateral to the DA depletion in an undrugged animal (thus, there is no obvious akinesia). In this study, we describe a method of examining behavior in unilateral 6-OHDA rats which addresses these concerns. When stepping behavior in the contralateral forelimb of unilateral 6-OHDA-treated rats was tested in isolation, severe and chronic akinesia was found in that limb. That is, when the hindquarters and ipsilateral forelimb were raised off the ground so that the contralateral forelimb alone supported the weight of the animal, the contralateral forelimb failed to step. Occasional trembling could be observed in the limb, but no more than a few small steps were ever initiated. In contrast, the isolated ipsilateral forelimb stepped normally (about 20 steps/min, which was similar to that of sham-operated animals when either limb was tested in isolation). After D-amphetamine (3 mg/kg, i.p.), which caused ipsiversive circling in a standard rotation test, the number of steps taken by the isolated ipsilateral limb in the 6-OHDA rats increased to a mean of 48 steps/min, which was similar to that of amphetamine-treated sham control rats in this test. However, amphetamine did not significantly increase the number of steps taken by the isolated contralateral limb, which remained aldnetic. After apomorphine (0.5 mg/kg, s.c.), which caused contraversive circling in a standard rotation test, the number of steps taken by the isolated ipsilateral limb in the 6-OHDA rats increased to a mean of 45 steps/min, which was not significantly greater than that of apomorphine-treated sham control rats. Surprisingly, like amphetamine above, apomorphine did not significantly increase the number of steps taken by the isolated contralateral limb. Instead the limb remained akinetic, with intermittent bursts of dyskinetic, in-place movements. How is it that both limbs step equally during standard rotation tests? Further examination of circling behavior indicated that the ipsilateral (unimpaired) forelimb shifted the weight of the animal to a new location in space, whereas the contralateral forelimb made only catch-up steps designed to re-establish support of the body’s weight. The catch-up step in the contralateral limb appears not to require DA because nonlesioned animals treated with high doses of a DA antagonist (e.g., 3 mg/kg haloperidol) can readily step with either limb if the experimenter slowly
117 citations
TL;DR: Investigating an alternative to permanent or temporary immunosuppression using a rat model with nerve transplants from Brown- Norway rat donors to bridge defects in the sciatic nerve of Lewis rat recipients finds the level of recovery is less than that of recipients of nerve allografts with immunos Puppression.
Abstract: Research involving nerve transplantation has shown that tissue rejection limits the neurologic recovery unless the host is immunosuppressed. This study investigates an alternative to permanent or temporary immunosuppression using a rat model with nerve transplants from Brown- Norway rat donors to bridge defects in the sciatic nerve of Lewis rat recipients as these two inbred strains differ at both major and minor histocompatibility loci.The specific aim of this study was to evaluate if predegenerated nerve grafts decreased the tissue rejection and improved the neurologic recovery of animals with allogenic nerve grafts to avoid the problems associated with either short- or long-term immunosuppression. The animals in the experimental groups received cyclosporin-A, predegencrated grafts, both, or neither. The predegenerated grafts were produced by division of the nerve three weeks prior to grafting to allow for Wallerian degeneration to occur. The outcome was assessed by measurements stressing functional recovery (sensory testing, gait analysis, joint flexion contracture), studies of muscle recovery (muscle weight and hydroxyproline concentration), and histologic studies (axonal counts and inflammatory reaction). The animals receiving the predegenerated grafts without cyclosporin did have an improved recovery (joint flexion contracture 35° ± 8 ° and hydroxyproline ratio 1.52 ± 0.16) as compared to the joint flexion contractures and hydroxyproline ratios of the allograft group of animals without either cyclosporin- A or pretreatment and the ungrafted control group (47° ± 18°, 1.68 ± 0.34, and 53° ±15° ,4.50 ± 0.27, respectively, p < 0.01). However, all the isograft groups and allograft groups with cyclosporin-A, regardless of whether the graft had been predegenerated or not, had greater neurologic recovery than the allograft group with predegenerated grafts but without cyclosporin-A by the same parameters (p < 0.01). Allograft groups with short-term immunosuppression with cyclosporin-A did as well as isograft groups, and isograft groups with predegenerated grafts did not do any better than isografts without pretreatment (p <0.01).
Clinical Relevance: Predegenerated nerve allografts will allow for greater neurologic recovery than standard nerve allografts avoiding the complications of immunosuppression, but the level of recovery is less than that of recipients of nerve allografts with immunosuppression. Nerve transplants would avoid the problems of neurologic deficits at the donor site and allow multiple large deficits to be treated easily.
29 citations
TL;DR: The results indicate that it is possible to use a small animal model to study normal and pathological phenomena oniate fetal human neural tissues and take advantage of the model to achieve HIV infectivity offetal human brain outside the human body.
Abstract: Successful xenografting of first trimester human fetal CNS tissue and retina has been reported in the literature. We wished to test the feasibility ofusing the anterior chamber ofthe rat eye to support the development of more mature human fetal xenografts. Here we report on the successful outcome of human brain and retinal transplants. Adult host rats immunosuppressed with cyclosporin A accepted these xenografts and supported their further development. Periodic examination of the host eyes using a direct ophthalmoscope or an ophthalmic slit lamp permitted direct visual monitoring of the health and growth of the transplants. Histologically it was possible to identify neuronal, macroglial, and microglial (macrophage) cell types within the grafts. Mitotic activity and histogenetic differentiation took place. Blood vessels filled with hematic cells were commonly present within the grafts. The walls of these vessels prevented the leakageofhorseradish peroxidase, suggesting the presence of a functional brain-blood barrier in the graft. These results indicate that it is possible to use a small animal model to study normal and pathological phenomena oniate fetal human neural tissues. Our group has already taken advantage of the model to achieve HIV infectivity offetal human brain outside the human body.
21 citations
TL;DR: It is concluded that matrices carrying negatively charged groups influence favorably the astrocytosis and the deposition of connective tissue, and that this approach represents a new avenue in attempting to modulate the brain scar formation.
Abstract: In previous studies, hyperporous synthetic hydrogels of poly(glyceryl methacrylate) or p(GMA), containing bioadhesive substrates of collagen, were implanted into rat cerebral tissue in order to provide systems of oriented guidance channels for directing the growth of the scar and axons /28/. In the present study, ionic p(GMA)- collagen hydrogels containing polar chemical groups, either basic amino groups or acidic carboxyl groups, were evaluated for their tolerance and their effects on the brain scarring response and axonal reactivity after long-term implantation in the cerebral cortex. In all animals, the implants were well tolerated. Although both types of gels influenced the astroglial reaction near the bioimplant, hydrogels carrying carboxyl groups had the strongest influence on the elongation, the direction and the organization of astrocytic processes so that a glial matrix could form in regions of the gel. Extracellular material (e.g. reticulin) was also deposited into the gels carrying carboxyl groups. Although cortical nerve fibers .in the surrounding tissue showed a regenerative response, extending onto or into the matrices, this behavior seemed to depend more on the organization of the .astrocytic scar imposed by the gel than on the type of gel. We conclude that matrices carrying negatively charged groups influence favorably the astrocytosis and the deposition of connective tissue, and that this approach represents a new avenue in attempting to modulate the brain scar formation.
18 citations
TL;DR: Encapsulation within an immobilization matrix allows manipulation of the internal environment, thereby providing the ability to pre-treat cells with various factors in a non-invasive manner, which may enhance long-term cellular viability.
Abstract: Immobilization of discrete cell clusters within a partially crosslinked matrix prevents reaggregation of primary tissues and may provide a means for long-term maintenance of encapsulated cells. Dissociated bovine adrenal chromaffin (BAC) cells were suspended throughout crosslinked polyanionic microspheres previously shown to be selectively permeable. Microcapsules approximately 500 µm in diameter were seeded with: 1) three different densities of BAC cells; and 2) BAC cells suspended in Matrigel® or coseeded with a genetically modified nerve growth factor (NGF)- releasing fibroblast cell line. Each group was analyzed in vitro at 1, 4 and 8 weeks for spontaneous and potassium-evoked release of catecholamines, and maintained in vitro for up to 12 weeks for morphological observations. Over time, release of norepinephrine (NE) and epinephrine (EPI) diminished, while dopamine (DA) remained constant from the monoseeded capsules. In the coseeded group, an increase in potassium-evoked release of DA was observed from 1 to 4 weeks, and remained at that level up to 8 weeks. Encapsulated chromaffin cells retained a rounded morphology typical of undifferentiated cells. Intact chromaffin cells with well preserved and abundant secretory granules were observed ultrastructurally after 4 weeks in vitro. Small neurites from the chromaffin cells in the coseeded group were observed at 4 weeks with light microscopy, and up to 12 weeks with electron microscopy. Under static incubation conditions, 1 mM D-amphetamine resulted in a significant increase in the output of NE and DA from the coseeded capsules 8 weeks postimplantation, as compared to microcapsules loaded with chromaffin cells alone. Encapsulation within an immobilization matrix allows manipulation of the internal environment, thereby providing the ability to pre-treat cells with various factors in a non-invasive manner, which may enhance long-term cellular viability.
14 citations
TL;DR: The evidence suggesting that gangliosides mediate functional recovery by minimizing primary or secondary cell loss or promoting the regeneration or sprouting of damaged central nerves subsequent to injury is focused on.
Abstract: Gangliosides are endogenous sialic acid containing glycospingolipids which are highly concentrated in the central nervous system. Although they were first characterized over 40 years ago, the function(s) played by this unique class of lipids remain largely unknown. Gangliosides have been suggested to play a prominent role in both normal and abnormal developmental processes. In addition, several lines of convergent evidence have indicated that gangliosides exert pronounced trophic effects following damage to peripheral and central nerves. Gangliosides have been shown to (1) enhance cell survival and outgrowth in cultured and developing neurons; (2) promote the regeneration of damaged peripheral and central nerves, and (3) facilitate behavioral recovery by altering the pattern, extent and persistence of the biochemical, morphological and behavioral changes induced by neural trauma. Little is known, however, concerning the neurobiological mechanisms which subserve the. behavioral protection afforded by ganglioside treatment. This review focuses on the evidence suggesting that gangliosides mediate functional recovery by minimizing primary or secondary cell loss or promoting the regeneration or sprouting of damaged central nerves subsequent to injury. An understanding of the mechanisms, by which gangliosides produce their effects may lead to the development of more efficacious and rational primary or adjunct pharmacological treatments for central nervous system disorders.
12 citations
TL;DR: The possibility was tested that peripheral nerve co-grafts would increase the functional effects of adrenal medulla grafts in a 6-hydroxydopamine-lesioned rat model and the results showed that in the animals with substantial numbers of surviving chromaffin cells, however, the Animals with sciatic nerve Co-Grafts showed greater decreases in rotational behavior as compared to the animalsWith adrenal Medulla grafting alone.
Abstract: Major limitations of adrenal medulla transplantation in animal models of Parkinson's disease have been the relatively small behavioral effects and the poor or inconsistent graft survival. Transplantation of fragments of sural nerve in combination with adrenal medulla has been reported to increase the survival of chromaffin cells in adrenal medulla grafts in primates. In the present study, the possibility was tested that peripheral nerve co-grafts would increase the functional effects of adrenal medulla grafts in a 6-hydroxydopamine-lesioned rat model. Animals received unilateral substantia nigra lesions, and subsequently received intraventricular grafts of adrenal medulla, sciatic nerve, adrenal medulla plus sciatic nerve, or sham grafts consisting of medium only. Functional effects of the grafts were tested using apomorphine-induced rotational behavior. The sciatic nerve co-grafts did not increase the survival of TH-immunoreactive chromaffin cells. The co-grafting treatment also did not augment the overall effect of adrenal medulla grafts on rotational behavior. In the animals with substantial numbers of surviving chromaffin cells, however, the animals with sciatic nerve co-grafts showed greater decreases in rotational behavior as compared to the animals with adrenal medulla grafts alone, even though the number of surviving cells was not increased.
9 citations
TL;DR: It is concluded that IMP was responsible for preventing injured CNS fibre regeneration and double-staining immunohistochemistry of normal and gliotic tissue with anti-IMP monoclonal antibodies together with glial and neuronal markers permitted the unequivocal definition of inhibitor presenting cells by confocal microscopy.
Abstract: Gliotic tissue is the major obstacle to axon regeneration after CNS injury. We designed tissue culture assays to search for molecules responsible for neurite outgrowth inhibition in gliotic tissue. All the inhibitory activity in injured brain tissue was located in a plasma membrane heparan-sulphate and condroitin-sulphate type-proteoglycan of apparent molecular weight 200kDalton.The proteoglycan core protein (apparent MW 48,000kD) was biologically inactive, whereas the glycosamine-glycan (GAG) chains accounted for the inhibitory activity. Because of its cell location and mode of induction, the inhibitor was called injured membrane proteoglycan, IMP. IMP prevented neurite outgrowth initiation when attached to the culture substrate and caused growth cone collapse when added in solution to neurons with already growing neurites. We concluded that IMP was responsible for preventing injured CNS fibre regeneration. Double-staining immunohistochemistry of normal and gliotic tissue with anti-IMP monoclonal antibodies together with glial and neuronal markers, permitted the unequivocal definition of inhibitor presenting cells by confocal microscopy. IMPimmunostaining in normal CNS was observed exclusively on neurons. However, after a lesion, immunostaining occurred primarily on intensely GFAP-positive reactive astrocytes, but not on OX-42 positive microglia.
8 citations
TL;DR: It is demonstrated that transplantation of the autologous cervical sympathetic ganglion into the brain ameliorated MPTP-induced parkinsonism in the monkey and this transplantation method was applied to parkinsonian patients.
Abstract: We have demonstrated that transplantation of the autologous cervical sympathetic ganglion into the brain ameliorated MPTP-induced parkinsonism in the monkey. According to the encouraging data from this animal model, we have applied this transplantation method to parkinsonian patients. Eight patients (45 59 years, 5 male and 3 female) underwent stereotactic transplantation of the aUtologous stellate ganglion into the unilateral caudate nucleus and putamen. Under local anesthesia a stereotactic frame was fixed to the patient’s head, and the frame and patient’s brain were simultaneously imaged to obtain the coordinates of the target points. Then the stellate ganglion was removed and cut into 100 pieces in a tissue culture solution. The small blocks of the ganglion were stereotaxically transplanted into the head of the caudate nucleus and anterior putamen. One week before transplantation, administration of L-dopa was stopped in all 8 patients. Preand postoperative states of the patients were carefully observed by a neurologist and were scored using the unified parkinsonism rating scale (UPRS), Hoehn & Yahr’s stage and four timed tests. Postoperative courses of the patients were encouraging. Seven out of 8 patients showed amelioration of parkinsonism, but one patient did not. Most patients showed amelioration of gait disturbance and bradykinesia. In contrast, tremor of the 7 patients seemed to be slightly aggravated 2 4 weeks after transplantation. No L-dopa administration was needed in these patients. During follow-up periods from 2 months to 1 year, the seven patients showed gradual and continuous improvement of parkinsonian symptoms, especially gait disturbance and bradykinesia. Unilateral transplantation resulted
8 citations
TL;DR: Animals that received the longest viable ribbon grafts showed a reversal of this effect back toward base line, whereas monkeys whose grafts left little or no surviving tissue showed no behavioral improvement.
Abstract: Small multiple “ribbon” autografts of intact adrenal medulla stereotaxically implanted at several sites throughout the striatum in longtailed macaques (Macaca fascicularis) have been shown to contain large amounts of viable glandular tissue as long as eight weeks after transplantation /15/. Variations of technique clearly influence viability/12/. All monkeys were maintained in specially adapted rotometer cages /30/ so that 24-hour measurements of activity and directional bias could be gathered. Lesions induced by intracerebral injection of 6-hydroxydopamine in the substantia nigra produced the expected chronic decrease in percentage of contralateral turning in most of the 24 subjects. Animals that received the longest viable ribbon grafts showed a reversal of this effect back toward base line, whereas monkeys whose grafts left little or no surviving tissue showed no behavioral improvement.
8 citations
TL;DR: This work studied the molecular composition of the neurotrophic/growth factor cocktail synthesized, stored and liberated by adrenomedullary chromaffi cells, which promotes their viability and neuritic growth in vitro.
Abstract: We studied the molecular composition of the neurotrophic/growth factor cocktail synthesized, stored and liberated by adrenomedullary chromaffi cells. Chromaffin cells have a dual capacity as models of the neuronal biochemical machinery and as therapeutic tools (implants) in several neural disorders, most notably Parkinson’s disease. Cultured bovine chromaffin cells release a proteinaceous material into their culture medium that, when applied to a wide range of different embryonic neurons from the CNS and PNS in vitro, promotes their viability and neuritic growth /1-3/. Stimulation of the chromaffin cells with cholinergic agonists or high K + enhances the amounts of the trophic mate
TL;DR: Carried out rodent neurografting studies, and performed adrenal medulla autotransplantation on three patients with severe Parkinson's disease.
Abstract: carried out rodent neurografting studies, we performed adrenal medulla autotransplantation on three patients with severe Parkinson's dis
TL;DR: A panel of antibodies was used to define MHC expression in the developing rat embryo to determine the clinical potential of neural transplantation based on donor cell expression of MHC antigens.
Abstract: The clinical potential of neural transplantation depends upon the feasibility of allogenenic or xenogeneic transplants, particularly of fetal tissue. This, in turn, is influenced by donor cell expression of MHC antigens. MHC expression in the developing CNS in situ had not been defined. Here, a panel of antibodies was used to define MHC expression in the developing rat embryo.
TL;DR: The development, and afferent and efferent innervation of striatal grafts implanted in longterm lesioned striatum were studied and compared with that observed in previous studies of grafts implant in non-lesioned or acutely lesioned host striatum.
Abstract: It is well known that fetal striatal tissue grafted into excitotoxically lesioned adult striatum can partially restore the behavioural/1/ and biochemical /3/ deficits, and establish anatomical connections with the host tissue/2,46/. Those previous studies were undertaken after grafting in acutely (usually 1 4 weeks before) lesioned striatum. Several factors change in the lesioned area after a long time of evolution. However, the capacity of survival and anatomical integration with the host of striatal grafts implanted in chronically lesioned striatum has not been investigated. In this study the development, and afferent and efferent innervation of striatal grafts implanted in longterm lesioned striatum were studied and compared with that observed in our previous studies of grafts implanted in non-lesioned or acutely lesioned host striatum/2,4-6/. A total of 46 female Sprague-Dawley rats received unilateral intrastriatal injections of ibotenic acid. Three to twenty months later 33 of these rats were grafted with rat or mouse fetal striatal tissue into the same area. The transplanted rats were killed at 7 days, 2 weeks (mouse-to-rat grafts only), 4 6 weeks, 8 weeks and 11 weeks (mouse-to-rat grafts only), 7 months’and 12 months post grafting (p,g). Seven rats were lesioned but not transplanted, and sacrificed 6-12 months after the lesion. Finally, 6 acutely (10 days) lesioned rats grafted with the same cell suspension as 8 of the chronically lesioned rats were used as a control group. DARPP-32-immunocytochemistry was used to differentiate the striatum-like and nonstriatum-like tissue-types in the grafts /6/. Tyrosine hydroxylase (TH) and serotonin (5HT) immunostaining was used to study the afferents from the substantia nigra and the mesencephalic raphe, respectively. Frontal cortical injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) were made to study the cortico-striatal afferents to the grafts /4/. Finally, a mouse specific neuronal marker (M6) was used in mouse-to-rat grafts to study the efferent projections to the host brain from the striatal grafts/5/. There was an important shrinkage of the lesioned striatum, and the centre of the lesion was composed of capsula interna bundles close to each other and separated by thin bridges of non-neuronal striatal tissue. The density of the host monoaminergic fibres (THand 5-HTpositive fibres) in the lesion-only areas or at the graft-host border was not much reduced, as compared with the acute-lesion controls. In contrast, the cortical innervation was much reduced in long-term lesioned striatum, at the same time that a dense network of PHA-L labelled corticostriatal fibres was observed in the contralateral striatum. The transplants grew to about 70-80% of the size obtained with control grafts placed into acute lesions, and developed characteristic striatum-like (i.e. DARPP-32-positive) patches. These patches could already be observed in the grafts 7 days after transplantation, i.e. as early as in grafts implanted in acute lesions. The grafts received a dense dopaminergic innervation, which was selective for the striatal-like areas, and began as early as in grafts implanted in acute lesions. The 5-HT-positive fibres also innervated the transplant following a pattern similar to that observed in grafts implanted into acute excitotoxic lesions. As the cortical innervation appeared much degenerated, the
TL;DR: A link between catecholamine secretion by transplanted cells and motor improvement using a rat rotational behavior model is suggested.
Abstract: Cultured human catecholaminergic and noncatecholaminergic donor cells were used in neural transplantation experiments in a rat model of Parkinson's disease. Using two different human catecholaminergic neuroblastoma cell lines, one control non-catecholaminergic neuroblastoma cell line, and one sham control (tissue culture medium), transplants were made into the striatum using a modified Ungerstedt hemiparkinsonian rat model. Significant decreases in apomorphine-induced rotational behavior were produced by two of three catecholaminergic cell lines. Grafted cells staining positively for tyrosine hydroxylase (TH) and catecholamine fluorescence indicated viable catecholamine activity in the two cell lines which produced reductions in rotational behavior. Catecholamine fluorescence was not detected in either of the two controls. These data suggest a link between catecholamine secretion by transplanted cells and motor improvement using a rat rotational behavior model.
TL;DR: In the present experiments involving 12 longtailed macaques, some grafts remained in medium 2.5 h before being transplanted, without apparent loss of viability, and a large number of separate grafts to be derived from one adrenal medulla.
Abstract: The robust survival of stereotaxic adrenal medullary autografts in monkey brain parenchyma depends heavily on technique/11/. One aspect of technique critical for clinical applications of CNS grafting is the problem of spreading treatment effects throughout large regions in a primate brain. Stereotaxic placement of a large number of grafts, which would address this problem, would require that later grafts retain the capacity for viability over a long period of time during the surgery, while earlier grafts are being made. In the present experiments involving 12 longtailed macaques (Macaca fascicularis), some grafts remained in medium 2.5 h before being transplanted, without apparent loss of viability.
TL;DR: Intracerebral transplantation of genetically engineered cells is a useful method for delivering recombinant growth factors to the brain and their effects on the survival and neuronal transdifferentiation of intrastriatal adrenal chromaffin cell co-grafts were evaluated in the unilateral 6-hydroxydopamine-lesioned rat.
Abstract: Intracerebral transplantation of genetically engineered cells is a useful method for delivering recombinant growth factors to the brain. Despite the clear experimental and therapeutic potential of this technique, relatively few cell types have been studied as targets for genetic manipulation and transplantation. Our interest has been to determine whether primary type I astrocytes, the normal support cell of the CNS, are suitable for this purpose. For these studies, type I astrocytes purified from newborn rat cerebral cortex were genetically modified in culture to constitutively express a mouse/3-NGF transgene, by infection with a replication-defective retroviral vector as previously described (Cunningham et al., Brain Res 561: 192-202). Infected astrocytes were found to release NGF into the culture medium at a rate that is > 10-fold higher than that of sister cultures of uninfected astrocytes (9.3+_2.9 pg/10 s cells/h vs. <0.8 pg NGF/10 cells/h). To evaluate the potential of these transgenic astrocytes to provide trophic support to brain grafts, their effects on the survival and neuronal transdifferentiation of intrastriatal adrenal chromaffin cell co-grafts were evaluated in the unilateral 6-hydroxydopamine-lesioned rat (an animal model of Parkinson's disease). NGF-producing transgenic astrocytes (AsN.8) were fluorescently labeled with DiI, mixed 1:1 with suspensions of postnatal day 12 adrenal chro
TL;DR: Intrastriatal implantation of autologous adrenal medulla/intercostal nerve cografts in 3 patients with advanced PD is performed and the early experience is reported here.
Abstract: Intrastriatal grafting of autologous adrenal medullary tissue has yielded modest benefits in Parkinson’s disease (PD) patients /1/. Parallel studies in non-human primates with experimental parkinsonism have shown that cografts of autologous adrenal medulla and peripheral nerve yield greater behavioral improvement and graft survival/1/. Hence, over the past year we have performed intrastriatal implantation of autologous adrenal medulla/intercostal nerve cografts in 3 patients with advanced PD and report our early experience here.
TL;DR: The upper age limit for survival of human embryonic dopaminergic suspension grafts correlates well with the period of development of the human nigrostriatal pathway.
Abstract: There is substantial variability worldwide in the donor ages utilized in nigral transplantation programs for the treatment of Parkinson’s disease. In multiple non-human species, it has been repeatedly demonstrated that optimal graft survival occurs when embryonic nigral neurons are harvested before the period of axonal extension, most likely due to axotomy and cell death, of older neurons/1; see 2,3/. Neurons which form the human substantia nigra develop within the ventricular zone primarily between post-ovulatory (PO) day 33 and 63 /2,5/. The nigrostriatal bundle begins to develop at approximately PO day 56/2/. From this data it could be predicted that the optimal period for suspension graft survival would occur before PO day 56 /2,3/. Solid grafts in nonhuman species have been found to have a slightly longer donor age \"window\" for graft harvesting than suspension grafts/see 3/. We compared the ability of solid and suspension grafts of human embryonic dopaminergic neurons at different embryonic ages to survive grafting into 6-OHDA lesioned immunosuppressed rats. Donor age was determined using the atlas of O’Rahilly and Mtiller/4, also see 2/. Suspension grafts survived best from PO day 37 to 56 with diminishing success through PO day 65. These implants displayed numerous healthy tyrosine-hydroxylase-immunoreactive (TH-ir) neurons which sent extensive neuritic processes into the host striatum. Conversely, suspension grafts of human embryonic dopaminergic neurons survived poorly when the donor age was greater than PO 65 days. In contrast to suspension grafts, solid implants of human ventral mesencephalic tissue displayed only modest survival of TH-ir neurons derived from younger donors. These grafts also displayed a limited host innervation. Good/ excellent survival of TH-ir neurons was observed in solid grafts employing embryonic donors between PO day 45 and 65. Only trace numbers of TH-ir neurons Were present within grafts derived from PO 72 tissue. In conclusion, the upper age limit for survival of human embryonic dopaminergic suspension grafts correlates well with the period of development of the human nigrostriatal pathway. Optimal donor ages for human nigral suspension grafts range from PO day 37 to 56 days. The donor age \"window\" for transplantation of solid human embryonic dopaminergic grafts appears to be extended by about nine days in comparison with suspension grafts. In younger age ranges (less than or equal to 41 days), intraparenchymal suspension nigral grafts survive better than solid grafts. The optimal donor age for intraparenchymal solid grafts ranges from PO day 4565. There appears to be a sharp fall-off in the
TL;DR: C cografts of adrenal medulla with pretransected peripheral nerve are performed and the survival of grafted chromaffin cells and the degree of host intrinsic DA fiber recovery are observed.
Abstract: It has been reported that adrenal medullary grafts enhance recovery of striatal dopamine (DA) fibers in MPTP-treated mice/1,2/. Since adrenal medullary chromaffin cells contain several trophic factors such as gangliosides and basic fibroblast growth factor, an increase in survivability of grafted chromaffin cells might enhance the degree of host DA fiber recovery. The content of nerve growth factor (NGF) in the distal stump of transected sciatic nerve increases more than twenty times one day after transection/3/. In this study, we performed cografts of adrenal medulla with pretransected peripheral nerve and observed the survival of grafted chromaffin cells and the degree of host intrinsic DA fiber recovery.
TL;DR: This research demonstrates, for the first time, organized, complex physiological function and its underlying neuronal microcircuitry in mammalian subretinal transplants in brain related to neurodegenerative diseases.
Abstract: There has been a rapid growth in research focused on retinal transplantation as a strategy for restoration of or rescue of function in retinas rendered afunctional by disease or trauma. This strategy has been partially stimulated by recent transplantation efforts in brain related to neurodegenerative diseases such as Parkinson’s, Huntington’s, and Alzheimer’s. These latter efforts have been hindered by a dearth of information regarding the specific properties of the inputs, intrinsic processing and outputs within the brain underlying these diseases. This is especially significant when attempting to study correlated functional and structural properties of the implanted tissue. In contrast to such studies, this research utilizes the retina, of which there is greater knowledge about the inputs (complex light patterns), intra-retinal processing and anatomical micro-circuitry, and the output (patterns of ganglion cell activity). Our research demonstrates, for the first time, organized, complex physiological function and its underlying neuronal microcircuitry in mammalian subretinal transplants.
TL;DR: The results confirm and extend previous reports on the recovery from motor deficits seen in rats with unilateral DA-depletion that can be provided by fetal nigral grafts reinnervating the dorsal portions of the striatum with tyrosine hydroxylase-immunoreactive neurons extending large fibres both within and outside the graft.
Abstract: Male Wistar rats were lesioned with 6hydroxydopamine in the right substantia nigra. Six weeks later motor disturbance was assessed by amphetamine-(5 mg/kg) induced rotation for 90 min. Ten/1 of cell suspension (300,000 cells) from the ventral mesencephalon of rat embryos or vehicle were injected into the denervated striatum of two groups: T II: transplant distributed in two sites of the same trace in dorsal striatum; T III: transplant distributed in three sites in a triangular fashion in the dorsal striatum. The amphetamine test was then repeated at 1, 3, 6, 12 and 23 weeks. The mean turns/min were reduced in the transplanted animals and showed significant differences at 6 weeks, while rotation was almost reduced to zero at 23 weeks. The recovery from the motor disturbance did not differ between groups T II and TIII at any time. Rats receiving vehicle did not show any improvement in motor asymmetry. These results confirm and extend previous reports on the recovery from motor deficits seen in rats with unilateral DA-depletion that can be provided by fetal nigral grafts reinnervating the dorsal portions of the striatum/1,2/. All groups showed a significant difference in the levels of dopamine (DA), dihydroxyphenacetic acid (DOPAC), and homovanillic acid (HVA) when compared with normal animals. An increment in DA, DOPAC and HVA levels in dorsal striatum was detected in groups T II and T III in comparison with the control group; however, a significant difference was found only between T III and the control group. A small increment of less than 1% of DA level in dorsal striatum was able to reverse the motor disturbance occurring after caudate-putamen denervation. DA, DOPAC and HVA levels in dorsal striatum tended to increase in group T III in comparison with group T II; however, there were no differences in behavioral recovery between these groups. This suggests that recovery from motor asymmetry may reveal neurotransmitter changes at very low levels. The higher neurotransmitter levels in the T III group could be induced by a higher cell survival rate or by an improved development of surviving cells as compared to the T II group. It is also possible that the three site transplants are more widely distributed so that metabolites are more easily detected. Immunocytochemical studies showed tyrosine hydroxylase-immunoreactive neurons extending large fibres both within and outside the graft.
TL;DR: Based on two primary motivating factors, namely the successful functional grafting of embryonic nervous tissue to brain in animal models of neurological disorders, and the need of new therapeutic alternatives for many patients with severe Parkinson's disease, this work aims to provide a novel and effective treatment for these disorders.
Abstract: Based on two primary motivating factors, namely the successful functional grafting of embryonic nervous tissue to brain in animal models of neurological disorders, and the need of new therapeutic alternatives for many patients with severe Parkinson's disease, a
TL;DR: This work is developing a cellular transplant therapy for PD based on the use of polymer encapsulation to immuno-isolate animal cells and has developed a semi-automated, computer-controlled version of the co-extrusion approach suitable for rapidly manufacturing integrally sealed capsules.
Abstract: Cellular transplantation is rapidly becoming recognized as a possible therapeutic approach in human neurodegenerative disorders such as Parkinson’s disease (PD). The transition from basic research to broad scale clinical application requires the clear demonstration of the safety of this technology. A milestone in this transition involves the identification of suitable cells for transplantation. The use of dopaminergic tissue derived from fetal or adrenal sources has been explored in transplantation as a possible longterm treatment for PD. In this approach, several issues remain unresolved. First there has been an inability to demonstrate long-term survival of transplanted tissue with consistent functional recovery in the clinical trials conducted to date. Furthermore, the availability of donor tissue, the amount of tissue needed to produce functional recovery, the safety of the cell source, and the ethical perception of using human fetal tissue remain significant obstacles to the use of these tissues. The use of non-human donor cells provides distinct advantages over human fetal cells as a source of therapeutically transplanted cells. We are developing a cellular transplant therapy for PD based on the use of polymer encapsulation to immuno-isolate animal cells. The cells we currently employ, PC12 cells, are an established tissue culture line initially isolated from a rat adrenomedullary chromaffin cell tumor. Transplantation of encapsulated PC12 cells provides specific safety advantages: the cells are rapidly destroyed by the immune system if they escape from immuno-isolation, the cells are free from microbial and viral infections, and the cells are grown under controlled conditions so that each lot of cells will be from uniform, defined growth stages. The key element to the successful application of this technology lies in the immunoisolation of the cells. Cells are immuno-isolated by surrounding them with a pe/’mselective polymer membrane that allows nutrients to enter the capsule so that the cells will survive and function. Additionally, the membrane is fabricated such that the molecular weight cutoff allows dopamine and other low molecular weight molecules to leave the capsule. Cellular encapsulation within polymeric hollow fibers can be achieved in several ways: cells can be hand-loaded into pre-formed hollow fibers or cells can be co-extruded with polymer so that a continuous polymeric fiber forms around the living cells. Both of these processes require additional steps in which fibers are cut to capsule size specifications and the ends of these capsules are sealed. We have additionally developed a semi-automated, computer-controlled version of the co-extrusion approach suitable for rapidly manufacturing integrally sealed capsules. This approach is rapid and its reproducibility and safety is assured through hands-off process validation. Immuno-isolated capsules prepared from polyacrylonitrile-vinyl chloride copolymer (PAN/PVC) using sterile fabrication processes are biocompatible following striatal implantation. Analysis of host tissue at the implant site using light and electron microscopy has shown that polymer capsules implanted into the rodent or primate striatum produce a mild gliotic reaction that rapidly diminishes over time. Inclusion of PC12 cells within the capsule does not affect the biocompatibility, suggesting that the encapsulated cells do not produce, or that the capsule retains, physiologically active levels of inflammatory or immune mediators.
TL;DR: The transplantation model in which immune responses against brain allografts are amplified in host peripheral lymphoid organs by systemic immunization with donor tissue suggests that some allografls do not induce T-cell mediated immune rejection responses, and finds that MHC class I immunoreactivity is usually more evident within grafts in the third ventricle, as compared to lateral Ventricle.
Abstract: The characteristics of immune responses to neuronal tissue grafts within the CNS are still obscure. To investigate these properties, we chose the transplantation model in which immune responses against brain allografts are amplified in host peripheral lymphoid organs by systemic immunization with donor tissue. We studied several combinations of host and donor rat strains. Rejection responses varied considerably between groups. Systemic sensitization with BN-RTln tissue results in the rejection of established BN-RTln neuronal allografls in the lateral ventricle of host F344-RTlI rats (Freed et al., Prog Brain Res 1988; 78: 233-241). Surprisingly, if the host and donor strains are reversed, similar sensitization-induced brain graft rejection does not occur (Poltorak and Freed, Ann Neurol 1991; 29: 377-388). A similar asymmetry was observed in another experiment, when BN-RTln donor grafts were severely rejected in LEW-RTII hosts, while LEW-RTII grafts in BN-RT1 hosts were not. Such results cannot be explained only in terms of genetic disparity between the donor and host strains. Despite complete genetic disparity, for some rat strain combinations, brain allografts can survive after systemic sensitization. Moreover, in the majority of strain combinations with the LEWRTII as the donor strain, systemic sensitization did not produce complete graft destruction, whereas for all allografls from BN-RTln donors, systemic sensitization induced severe immune responses in the host animals. Therefore, immunogenicity of the donor tissue is an important factor involved in neural graft rejection, but additional factors, including Ir gene, host MHC allele and host susceptibility to autoimmune disease, may also be important. It has been suggested that grafted neuronal tissue demonstrates increased expression of MHC class I antigen, and that this enhancement is a crucial event leading to graft rejection. However, damage of the host brain during implantation procedures and injury of the grafted tissue itself also induces increased MHC class I antigen expression. Moreover, upregulation of MHC class II antigen expression in microglial cells, which can function as potential antigenpresenting cells, can be due to non-specific damage of host or donor tissue. Interestingly, we found that MHC class I immunoreactivity is usually more evident within grafts in the third ventricle, as compared to lateral ventricle. Increased expression of both MHC class I and II antigens in the grafted tissue did not predict allograft rejection. In certain rat strain combinations, allografts expressed increased MHC class I and II immunoreactivity without apparent immunological rejection responses and without lymphocytic infiltration. Therefore, it seems that simple upregulation of MHC antigen expression within the grafted tissue is not sufficient to induce a brain allograft rejection response. After host sensitization with donor tissue, allografts showed increased cell density, as compared to sham-sensitized allografts. Nevertheless, in some rat strain combinations, we were not able to detect T cell infiltration within the allografted tissue. In these cases, the infiltrating cells consisted primarily of activated microglial cells and blood-derived macrophages. These data support the hypothesis that some allografls do not induce T-cell mediated immune rejection responses. This type of reaction would be mediated primarily by microglial and macrophage cytokines. However, such immune responses are
TL;DR: The living organ donor framework is more respectful and protective of the woman who is asked to donate fetal tissue than the cadaver donor framework and will ensure that both the woman and the fetus are protected and respected.
Abstract: Treating the aborted fetus as a human cadaver is believed to be justified because it is respectful and protective of the fetus. In the United States, most states permit the donation and procurement of human fetal tissue for educational, research, or therapeutic purposes in accord with the Uniform Anatomical Gift Act (UAGA). These state laws specify the protections necessary for the fetal cadaver and the circumstances under which the next-of-kin may donate its remains. However, treating the woman who agrees to donate fetal tissue merely as one of the next-of-kin who may consent to donate the tissue of her deceased relative is disrespectful and leaves her vulnerable to harm. It overlooks both the risks that donating fetal tissue pose to her as well as the unique relationship that exists between a woman and a fetus. The cadaver donor framework, for example, does not require the woman’s consent before fetal tissue may be used; the consent of the father is considered sufficient. And, in any case, only minimal information is required to be disclosed to those deciding whether to donate the tissue of a deceased relative. The following are possible risks to a woman’s privacy and well-being associated with donating tissue for transplantation after elective abortion: Her blood may be tested for infectious agents such as HIV. The tissue may be labeled with an identifier that makes it possible to trace the woman who had the abortion and donated the tissue. Persons uninvolved in the woman’s care may have access to her clinic records to obtain information about her medical history. The abortion procedure may be modified in ways that prolong or intensify discomfort, or that increase the risks of physical harm to the woman. The living organ donor framework is more respectful and protective of the woman who is asked to donate fetal tissue than the cadaver donor framework. It acknowledges that the woman has a unique and privileged relationship with the fetus and that donation poses a variety of significant risks to the woman. Her respect and protection should be secured in the following ways: Her consent should be necessary; the father’s consent should never be sufficient. She should be informed of the proposed use of the tissue. She should be informed of the risks that donation poses to her privacy. She should be informed of the risks associated with testing her blood or urine to establish the quality of the tissue, labeling the fetal tissue with an identifier traceable to her, and tissue procurers or others not directly involved in her care gaining access to private information in her clinic record. She should be informed of any modifications of the abortion procedure and the associated risks to her well-being. She should be informed of the risks of prolonged or intensified discomfort as well as the risks of incomplete abortion or trauma to the cervix associated with any modifications, e.g., slowing the abortion procedure, decreasing the pressure of the suction, or increasing the dilatation of the cervix and the cannula. Neither the cadaver donor framework, nor the living organ donor framework, by itself captures the full range of obligations to both the fetus and the woman involved in fetal tissue transplantation. Combining these frameworks, however, so that they complement and constrain, rather than conflict with one another, will ensure that both the woman and the fetus are protected and respected.
TL;DR: In vitro binding of [3H]- estradiol to cytosolic preparations provided evidence of estrogen receptors within the transplants, and high levels correspond to previous work demonstrating elevated estrogen receptor levels during the first postnatal week in the rat cerebral cortex.
Abstract: Fetal neocortical tissue was grafted into neocortical lesion cavities made in newborn rats. After two weeks survival, in vitro binding of [H3]- estradiol to cytosolic preparations provided evidence of estrogen receptors within the transplants. The observed high levels correspond to previous work demonstrating elevated estrogen receptor levels during the first postnatal week in the rat cerebral cortex.
TL;DR: The use of the non-viral, calcium phosphate transfection method to introduce stably expressed genes into primary cultures of striatal astrocytes for subsequent use in brain grafting is reported here and the regulation of gene product expression by systemically administered drugs is introduced.
Abstract: In the late 1980s, several investigators applied the strategy of combining genetic engineering with brain grafting (for review see Gage et al., Neuron 1991; 6: 1). Disadvantages associated with current approaches include the use of non-brain donor tissue (fibroblasts), the use of tumor-derived cell lines, and the use of viral infection methods to introduce the gene of interest. We report here the use of the non-viral, calcium phosphate transfection method to introduce stably expressed genes into primary cultures of striatal astrocytes for subsequent use in brain grafting. Astrocytes may be the preferred cellular vehicle for gene replacement therapy in brain transplantation paradigms because they divide in culture, are brain-derived and regionally specific, possess a release mechanism , secrete growth factors, can migrate several millimeters from the transplant site, and have neurotransmitter receptors. In addition, we introduce and demonstrate the regulation of gene product expression by systemically administered drugs. Thus, by the incorporation of inducible promoters in the transfected gene sequence, drugs which act upon astrocyte neuro-transmitter receptors can regulate the level of transfected gene expression. Neonatal (day 2) rat striatal astrocytes were transfected with RSV-driven or enkephalin promoter-driven chloramphenicol acetyl trans-ferase (CAT) chimeric plasmids after 3 weeks in culture. In addition, the incorporation of the neomycin resistance gene combined with selective growth of transfected cells in G418-containing (300 /sg/ml) media for 3 additional weeks insured stable transfected gene incorporation from all surviving astrocytes. These cultured astrocytes expressed CAT enzymatic activity , which was measured both in cell lysates and in the conditioned media. As the cells in these cultures were 100% viable, the latter observation indicates that the astrocytes secreted CAT into the media. Following trans-plantation into the adult rat striatum, the transplanted astrocytes were identified histologi-cally by astrocyte-specific GFAP staining, as well as with antibody staining to CAT protein to specifically detect the transfected cells. To be certain that GFAP and CAT staining identified transplanted cells, CAT activity was also biochemically detected in tissue blocks containing the transplant at both 1 day and 3 weeks after transplantation.