The boundlessness of behavioral neuroscience: A look across 30 years.

It has been more than thirty years since the two inaugural IBNS presidents sat down at a larger neuroscience conference and decided that there should be more to behavioral neuroscience than a single theme at a meeting. The progeny of these conversations is the International Behavioral Neuroscience Society (IBNS) and this year will be its thirty year anniversary. We reflect back on the last thirty years of the research career of the society's second president, Paul R. Sanberg, as an example of how behavioral neuroscience research has changed these last few decades.

Specific antibody binding to the APP672-699 region shifts APP processing from α- to ß-cleavage.

Alzheimer's disease (AD), a progressive neurodegenerative disorder that is the most common cause of dementia in the elderly, is characterized by the accumulation of amyloid-ß (Aß) plaques and neurofibrillary tangles, as well as a progressive loss of synapses and neurons in the brain. The major pertinacious component of amyloid plaques is Aß, a variably sized peptide derived from the integral membrane protein amyloid precursor protein (APP). The Aß region of APP locates partly within its ecto- and trans-membrane domains. APP is cleaved by three proteases, designated as α-, ß-, and γ-secretases. Processing by ß- and γ-secretase cleaves the N- and C-terminal ends of the Aß region, respectively, releasing Aß, whereas α-secretase cleaves within the Aß sequence, releasing soluble APPα (sAPPα). The γ-secretase cleaves at several adjacent sites to yield Aß species containing 39-43 amino acid residues. Both α- and ß-cleavage sites of human wild-type APP are located in APP672-699 region (ectodomain of ß-C-terminal fragment, ED-ß-CTF or ED-C99). Therefore, the amino acid residues within or near this region are definitely pivotal for human wild-type APP function and processing. Here, we report that one ED-C99-specific monoclonal antibody (mAbED-C99) blocks human wild-type APP endocytosis and shifts its processing from α- to ß-cleavage, as evidenced by elevated accumulation of cell surface full-length APP and ß-CTF together with reduced sAPPα and α-CTF levels. Moreover, mAbED-C99 enhances the interactions of APP with cholesterol. Consistently, intracerebroventricular injection of mAbED-C99 to human wild-type APP transgenic mice markedly increases membrane-associated ß-CTF. All these findings suggest that APP672-699 region is critical for human wild-type APP processing and may provide new clues for the pathogenesis of sporadic AD.

Stem cells and G-CSF for treating neuroinflammation in traumatic brain injury: aging as a comorbidity factor.

Traumatic brain injury (TBI), often called the signature wound of Iraq and Afghanistan wars, is characterized by a progressive histopathology and long-lasting behavioral deficits. Treatment options for TBI are limited and patients are usually relegated to rehabilitation therapy and a handful of experimental treatments. Stem cell-based therapies offer alternative treatment regimens for TBI, and have been intended to target the delayed therapeutic window post-TBI, in order to promote "neuroregeneration," in lieu of "neuroprotection" which can be accomplished during acute TBI phase. However, these interventions may require adjunctive pharmacological treatments especially when aging is considered as a comorbidity factor for post-TBI health outcomes. Here, we put forward the concept that a combination therapy of human umbilical cord blood cell (hUCB) and granulocyte-colony stimulating factor (G-CSF) attenuates neuroinflammation in TBI, in view of the safety and efficacy profiles of hUCB and G-CSF, their respective mechanisms of action, and efficacy of hUCB+G-CSF combination therapy in TBI animal models. Further investigations on the neuroinflammatory pathway as a key pathological hallmark in acute and chronic TBI and also as a major therapeutic target of hUCB+G-CSF are warranted in order to optimize the translation of this combination therapy in the clinic.

Monocytes are essential for the neuroprotective effect of human cord blood cells following middle cerebral artery occlusion in rat.

Systemic administration of human umbilical cord blood (HUCB) mononuclear cells (MNC) following middle cerebral artery occlusion (MCAO) in the rat reduces infarct size and, more importantly, restores motor function. The HUCB cell preparation is composed of immature T-cells, B-cells, monocytes and stem cells. In this study we examined whether the beneficial effects of HUCB injection were attributable to one of these cell types. Male Sprague Dawley rats underwent permanent MCAO followed 48 h later by intravenous administration of HUCB MNC preparations depleted of either CD14(+) monocytes, CD133(+) stem cells, CD2(+) T-cells or CD19(+) B cells. Motor function was measured prior to MCAO and 30 days post-stroke. When CD14(+) monocytes were depleted from the HUCB MNC, activity and motor asymmetry were similar to the MCAO only treated animals. Monocyte depletion prevented HUCB cell treatment from reducing infarct size while monocyte enrichment was sufficient to reduce infarct size. Administration of monocyte-depleted HUCB cells did not suppress Iba1 labeling of microglia in the infarcted area relative to treatment with the whole HUCB preparation. These data demonstrate that the HUCB monocytes provide the majority of the efficacy in reducing infarct volume and promoting functional recovery.

Permeating the blood brain barrier and abrogating the inflammation in stroke: implications for stroke therapy.

Cell therapy has been shown as a potential treatment for stroke and other neurological disorders. Human umbilical cord blood (HUCB) may be a promising source of stem cells for cell therapy. The most desired outcomes occur when stem cells cross the blood brain barrier (BBB) and eventually reach the injured brain site. We propose, from our previous studies, that mannitol is capable of disrupting the BBB, allowing the transplanted cells to enter the brain from the periphery. However, when the BBB is compromised, the inflammatory response from circulation may also be able to penetrate the brain and thus may actually exacerbate the stroke rather than afford therapeutic effects. We discuss how an NF-kB decoy can inhibit the inflammatory responses in the stroke brain thereby reducing the negative effects associated with BBB disruption. In this review, we propose the combination of mannitol-induced BBB permeation and NF-kB decoy for enhancing the therapeutic benefits of cell therapy in stroke.

Human umbilical cord blood cells alter blood and spleen cell populations after stroke.

The human umbilical cord blood (HUCB) mononuclear cell (MNC) fraction is a mixed population of cells that induces functional repair in rodent models of stroke when injected intravenously (i.v.). The transplanted cells are found in the infarcted hemisphere and the spleen. The goal of this project was to determine the nature of the interaction between the HUCB MNCs cells and splenic immune cells. Male Sprague Dawley rats underwent permanent middle cerebral artery occlusion (MCAO) and received i.v. injection of either vehicle (MCAO only), HUCB MNCs or MNCs depleted of CD14+ monocytes, CD133+ stem cells or CD19+ B cells 48 hours post-stroke. At 72 hours post-MCAO, the animals were euthanized and the spleens and blood MNCs harvested for flow cytometry and mitogen proliferation assays. All HUCB cell preparations decreased the percentage of T cells in the spleen and monocytes in the blood (p < 0.05). MNCs depleted of CD14+ and CD19+ decreased the percentage of macrophage (p < 0.001), while CD133 depleted MNCs increased the percentage of macrophage in spleen (p < 0.001); MNC did not alter the macrophage population from the level observed after MCAO. Only HUCB MNC significantly decreased Concanavalin A (ConA)-induced T cell stimulation (p < 0.05). These results suggest that the effects of HUCB MNC in the spleen are not due to a single HUCB population, but the interaction of all the subpopulations together.

NT-020, a natural therapeutic approach to optimize spatial memory performance and increase neural progenitor cell proliferation and decrease inflammation in the aged rat.

The process of aging is linked to oxidative stress, microglial activation, and proinflammatory factors, which are known to decrease cell proliferation and limit neuroplasticity. These factors may lead the transition from normal aging to more severe cognitive dysfunction associated with neurodegenerative diseases. We have shown that natural compounds such as polyphenols from blueberry and green tea and amino acids like carnosine are high in antioxidant and antiinflammatory activity that decreases the damaging effects of reactive oxygen species (ROS), in the blood, brain, and other tissues of the body. Furthermore, we have shown that the combination of these nutrients (called NT-020) creates a synergistic effect that promotes the proliferation of stem cells in vitro and in vivo. In the current study, we examined the effects of NT-020 on neurogenesis and performance on a Morris water maze (MWM). Aged (20-month-old) male Fischer 344 rats were treated with 135.0 mg/kg per day (n = 13) of NT-020. Young (3-month-old) (n = 10) and aged (20-month-old) (n = 13) control male Fischer 344 rats were treated with water by oral gavage. All groups were treated for a period of 4 weeks. Although there was no difference in performance in the MWM when comparing all aged rats, when the data for aged impaired rats were compared, there was a significant difference between groups on the last day of training with the treatment group performing better than controls. Using the cell cycle-regulating protein (Ki67), doublecortin (DCX), and OX6 antibody markers, cell proliferation, neurogenesis, and microglial activation were estimated in the dentate gyrus (DG) of young and aged animals. Cell proliferation was also examined in the subventricular zone (SVZ). A decreased number of OX6 MHC II-positive cells, increased neurogenesis, and increased number of proliferating cells were found in rats treated with NT-020 in comparison with aged control rats. In sum, NT-020 may promote health, proliferation, and maintenance of neurons in the age animals and exert antiinflammatory actions that promote function in the aged stem cell niche.

Mannitol facilitates neurotrophic factor up-regulation and behavioural recovery in neonatal hypoxic-ischaemic rats with human umbilical cord blood grafts.

We recently demonstrated that blood-brain barrier permeabilization using mannitol enhances the therapeutic efficacy of systemically administered human umbilical cord blood (HUCB) by facilitating the entry of neurotrophic factors from the periphery into the adult stroke brain. Here, we examined whether the same blood-brain barrier manipulation approach increases the therapeutic effects of intravenously delivered HUCB in a neonatal hypoxic-ischaemic (HI) injury model. Seven-day-old Sprague-Dawley rats were subjected to unilateral HI injury and then at day 7 after the insult, animals intravenously received vehicle alone, mannitol alone, HUCB cells (15k mononuclear fraction) alone or a combination of mannitol and HUCB cells. Behavioural tests at post-transplantation days 7 and 14 showed that HI animals that received HUCB cells alone or when combined with mannitol were significantly less impaired in motor asymmetry and motor coordination compared with those that received vehicle alone or mannitol alone. Brain tissues from a separate animal cohort from the four treatment conditions were processed for enzyme-linked immunosorbent assay at day 3 post-transplantation, and revealed elevated levels of GDNF, NGF and BDNF in those that received HUCB cells alone or when combined with mannitol compared with those that received vehicle or mannitol alone, with the combined HUCB cells and mannitol exhibiting the most robust neurotropic factor up-regulation. Histological assays revealed only sporadic detection of HUCB cells, suggesting that the trophic factor-mediated mechanism, rather than cell replacement per se, principally contributed to the behavioural improvement. These findings extend the utility of blood-brain barrier permeabilization in facilitating cell therapy for treating neonatal HI injury.

Neural transplants in patients with Huntington's disease undergo disease-like neuronal degeneration.

The clinical evaluation of neural transplantation as a potential treatment for Huntington's disease (HD) was initiated in an attempt to replace lost neurons and improve patient outcomes. Two of 3 patients with HD reported here, who underwent neural transplantation containing striatal anlagen in the striatum a decade earlier, have demonstrated marginal and transient clinical benefits. Their brains were evaluated immunohistochemically and with electron microscopy for markers of projection neurons and interneurons, inflammatory cells, abnormal huntingtin protein, and host-derived connectivity. Surviving grafts were identified bilaterally in 2 of the subjects and displayed classic striatal projection neurons and interneurons. Genetic markers of HD were not expressed within the graft. Here we report in patients with HD that (i) graft survival is attenuated long-term; (ii) grafts undergo disease-like neuronal degeneration with a preferential loss of projection neurons in comparison to interneurons; (iii) immunologically unrelated cells degenerate more rapidly than the patient's neurons, particularly the projection neuron subtype; (iv) graft survival is attenuated in the caudate in comparison to the putamen in HD; (v) glutamatergic cortical neurons project to transplanted striatal neurons; and (vi) microglial inflammatory changes in the grafts specifically target the neuronal components of the grafts. These results, when combined, raise uncertainty about this potential therapeutic approach for the treatment of HD. However, these observations provide new opportunities to investigate the underlying mechanisms involved in HD, as well as to explore additional therapeutic paradigms.

Effects of Sertoli cell-conditioned medium on ventral midbrain neural stem cells: a preliminary report.

The 796RMB cell line is a multipotent stem cell line isolated from human fetal midbrain tissues, a region from which dopamine neurons of the substantia nigra develop. It would be useful to increase the dopaminergic characteristics of this cell line to enhance its usefulness as a cell therapy for Parkinson's disease utilizing transplantation protocols. Sertoli cells and its conditioned media isolated from the testis have been previously shown to enhance tyrosine hydroxylase expression in ventral mesencephalon neurons both in vitro and in vivo. Therefore, the present preliminary study investigated the ability of Sertoli cell pre-conditioned medium to enhance differentiation of the 796MB cell line toward the domaminergic phenotype. Results showed that secretory products derived from Sertoli cell conditioned medium increased cell proliferation and enhanced dopaminergic neuronal differentiation of the 796RMB cell line. These findings may lead to alternative therapeutic cell transplantation protocols for the treatment of Parkinson's disease.

A comparison of dopaminergic cells from the human NTera2/D1 cell line transplanted into the hemiparkinsonian rat.

Human NT cells derived from the NTera2/D1 cell line express a dopaminergic phenotype making them an attractive vehicle to supply dopamine to the depleted striatum of the Parkinsonian patient. In vitro, hNT neurons express tyrosine hydroxylase (TH), depending on the length of time they are exposed to retinoic acid. This study compared two populations of hNT neurons that exhibit a high yield of TH+ cells, MI-hNT and DA-hNT. The MI-hNT and DA-hNT neurons were intrastriatally transplanted into the 6-OHDA hemiparkinsonian rat. Amelioration in rotational behavior was measured and immunohistochemistry was performed to identify surviving hNT and TH+ hNT neurons. Results indicated that both MI-hNT and DA-hNT neurons can survive in the striatum, however, neither maintained their dopaminergic phenotype in vivo. Other strategies used in conjunction with hNT cell replacement are likely needed to enhance and maintain the dopamine expression in the grafted cells.

Sertoli cells induce systemic donor-specific tolerance in xenogenic transplantation model.

Cell therapy is a potentially powerful tool in the treatment of many grave disorders including leukemia, immune deficiencies, autoimmune diseases, and diabetes. However, finding matched donors is challenging and recipients may suffer from the severe complications of systemic immune suppression. Sertoli cells, when cotransplanted with both allo- and xenograft tissues, promote graft acceptance in the absence of systemic immunosuppression. How Sertoli cells do this is not, as yet, clearly defined. We have examined the ability of Sertoli cells to produce systemic immune tolerance. For this purpose, Sertoli cells were injected into an otherwise normal C57/BL6 mouse host via the lateral tail vein. No other immunosuppressive protocols were applied. Six to 8 weeks posttransplantation, blood was collected for analysis of cytokine levels. Tolerance to donor cells was determined by mixed lymphocytic culture, and production of T-cell-dependent antibody was determined by an in vitro anti-sheep red blood cell plaque-forming assay. Results showed a marked modulation of immune cytokines in the transplanted mouse host and donor-specific transplantation tolerance was achieved. Tolerant mouse lymphocytes maintained a competent humoral antibody response. Additionally, C57/BL6 mice transplanted with rat Sertoli cells tolerated rat skin grafts significantly longer than control non-Sertoli cell transplanted mice. We conclude that systemic administration of rat Sertoli cells across xenogenic barrier induces transplantation tolerance without altering systemic immune competence. These data suggest that Sertoli cells may be used as a novel and potentially powerful tool in cell transplantation therapy.

Influence of retinoic acid and lithium on proliferation and dopaminergic potential of human NT2 cells.

Our laboratory is working with the human NTera2/D1 (NT2) cell line, which has properties similar to those of progenitor cells in the central nervous system (CNS). These neural-like precursor cells can differentiate into all three major lineages, neurons, astrocytes, and oligodendrocytes. The pure neuronal population, hNT neurons, possess characteristics of dopamine (DA) cells. First, we analyzed whether the retinoic acid (RA)-treated hNT neurons and the NT2 precursor cells expressed two transcription factors required for development of the midbrain DA neurons. We report that NT2 cells endogenously expressed Engrailed-1 and Ptx3, whereas RA-treated hNT neurons did not express Engrailed-1 or Ptx3. Next we examined the influence of lithium treatment on Engrailed-1 and Ptx3 as well as another critical transcription factor, Nurr1. Previous research has shown that lithium can mimic the Wnt pathway, which is important for the induction of these transcription factors. Finally, we investigated the effect of lithium treatment on the viability and proliferation of NT2 cells, because lithium has been shown to stimulate neurogenesis in adult neural precursors. Lithium treatment increased the viability and proliferation of NT2 cells. The expression of transcription factors essential for the induction and maintenance of the DA phenotype was not increased in NT2 after lithium treatment. We conclude that the NT2 cell line is an excellent in vitro model system for studying the influence of pharmalogical agents on proliferation, differentiation, and apoptosis of a human neural progenitor cell line.

Survival of rat or mouse ventral mesencephalon neurons after cotransplantation with rat sertoli cells in the mouse striatum.

Transplanting cells across species (xenotransplantation) for the treatment of Parkinson's disease has been considered an option to alleviate ethical concerns and shortage of tissues. However, using this approach leads to decreased cell survival; the xenografted cells are often rejected. Sertoli cells (SCs) are testis-derived cells that provide immunological protection to developing germ cells and can enhance survival of both allografted and xenografted cells. It is not clear whether these cells will maintain their immunosuppressive support of cografted cells if they are transplanted across species. In this study, we investigated the immune modulatory capacity of SCs and the feasibility of xenografting these cells alone or with allografted and xenografted neural tissue. Transplanting xenografts of rat SCs into the mouse striatum with either rat or mouse ventral mesencephalon prevented astrocytic infiltration of the graft site, although all transplants showed activated microglia within the core of the graft. Surviving tyrosine hydroxylase-positive neurons were observed in all conditions, but the size of the grafts was small at best. SCs were found at 1 and 2 weeks posttransplant. However, few SCs were found at 2 months posttransplant. Further investigation is under way to characterize the immune capabilities of SCs in a xenogeneic environment.

Behavioral alterations in Lewis rats following two-day continuous 3-nitropropionic acid administration.

The mitochondrial toxin, 3-nitropropionic acid (3-NP), produces motor dysfunction and striatal atrophy in rats. However, rat strain and method of administration may contribute to variability in the deficits caused by 3-NP toxicity. To evaluate this, changes in nocturnal spontaneous locomotor activity from chronic administration of 3-NP using an osmotic mini pump, were examined in the Lewis rats. Lewis rats were treated with 3-NP or saline for 2 days and behavior was tested daily for a 15 day period. Animals receiving 3-NP displayed significantly less spontaneous activity than animals in the saline group. 3-NP treated animals also weighed significantly less when compared to saline treated animals. These results demonstrate that even though there were no significant alterations in overt anatomical pathology, even short-term exposure to 3-NP produced significant effects. This short-term administration may present a potential paradigm for examination of sub-threshold neurotoxicity.

Do hematopoietic cells exposed to a neurogenic environment mimic properties of endogenous neural precursors?

Hematopoietic progenitors are cells, which under challenging experimental conditions can develop unusual phenotypic properties, rather distant from their original mesodermal origin. As previously reported, cells derived from human umbilical cord blood (HUCB) or human bone marrow (BM) under certain in vivo or in vitro conditions can manifest neural features that resemble features of neural-derived cells, immunocytochemically and in some instances also morphologically. The present study explored how hematopoietic-derived cells would respond to neurogenic signals from the subventricular zone (SVZ) of adult and aged (6 and 16 months old) rats. The mononuclear fraction of HUCB cells was transplanted into the SVZ of immunosuppressed (single cyclosporin or three-drug treatment) animals. The triple-suppression paradigm allowed us to protect transplanted human cells within the brain and to explore further their phenotypic and migratory properties. One week after implantation, many surviving HUCB cells were located within the SVZ and the vertical limb of the rostral migratory stream (RMS). The migration of HUCB cells was restricted exclusively to the pathway leading to the olfactory bulb. In younger animals, grafted cells navigated almost halfway through the vertical limb, whereas, in the older animals, the migration was less pronounced. The overall cell survival was greater in younger animals than in older ones. Immunocytochemistry for surface CD antigen expression showed that many HUCB cells, either cultured or within the brain parenchyma, retained their hematopoietic identity. A few cells, identified by using human-specific antibodies (anti-human nuclei, or mitochondria) expressed nestin and doublecortin, markers of endogenous neural progenitors. Therefore, it is believed that the environment of the neurogenic SVZ, even in aged animals, was able to support survival, "neuralization," and migratory features of HUCB-derived cells.

Formation and structure of transplantable tissue constructs generated in simulated microgravity from Sertoli cells and neuron precursors.

Cell transplantation therapy for Parkinson's disease (PD) has received much attention as a potential treatment protocol for this neurodegenerative condition. Although there have been promising successes with this approach, it remains problematic, especially regarding the inability to provide immediate trophic support to the newly grafted cells and the inability to prevent acute and/or long-term graft rejection by the host. To address these issues of cell graftability, we have created a novel tissue construct from isolated rat Sertoli cells (SC) and the NTerra-2 immortalized human neuron precursor cell line (NT2) utilizing NASA-developed simulated microgravity technology. The two cell types were cocultured at a 1:4 (SC/NT2) ratio in the High Aspect Rotating Vessel (HARV) biochamber for 3 days, after which a disc-shaped aggregate (1-4 mm diameter) was formed. Sertoli neuron aggregated cells (SNAC) were collected by gravity sedimentation and processed either for light and electron microscopy or for fluorescent immunocytochemistry. Intra-SNAC clusters of SC and NT2 cells were identified by anti-human mitochondrial protein (huMT--specific for NT2 cells) and cholera toxin subunit B (CTb--specific for SC). There was little evidence of cell death throughout the aggregate and the absence of central necrosis, as might be expected in such a large aggregate in vitro. Ultrastructurally, SC did not express junctional modifications with NT2 cells nor with adjacent SC as is typical of SC in vivo and, in some protocols, in vitro. NT2 cells, however, showed distinct intercellular junction-like densities with adjacent NT2 cells, often defining canaliculi-like channels between the microvillus borders of the cells. The results show that the use of simulated microgravity coculture provides a culture environment suitable for the formation of a unique and viable Sertoli-NT2 (i.e., SNAC) tissue construct displaying intra-aggregate cellular organization. The structural integration of SC with NT2 cells provides a novel transplantable tissue source, which can be tested to determine if SC will suppress rejection of the grafted NT2 cells and provide for their short- and long-term trophic support in situ in the treatment of experimental PD.

Intravenous versus intrastriatal cord blood administration in a rodent model of stroke.

Human umbilical cord blood (hUCB) is a rich source of hematopoietic stem cells that have been used to reconstitute immune cells and blood lineages. Cells from another hematopoietic source, bone marrow, have been found to differentiate into neural cells and are effective in the treatment of stroke. In this study, we administered hUCB cells intravenously into the femoral vein or directly into the striatum and assessed which route of cell administration produced the greatest behavioral recovery in rats with permanent middle cerebral artery occlusion (MCAO). All animals were immunosuppressed with cyclosporine (CSA). When spontaneous activity was measured using the Digiscan automated system, it was found to be significantly less when hUCB was transplanted 24 hr after stroke compared with nontransplanted, stroked animals (P < 0.01). Furthermore, behavioral recovery was similar with both striatal and femoral hUCB delivery. This is in contrast to the step test, in which significant improvements were found only after femoral delivery of the hUCB cells. In the passive avoidance test, transplanted animals learned the task faster than nontransplanted animals (P < 0.05). Together, these results suggest that hUCB transplantation may be an effective treatment for brain injuries, such as stroke, or neurodegenerative disorders. In addition, intravenous delivery may be more effective than striatal delivery in producing long-term functional benefits to the stroked animal.

Reconstructive neurosurgery for Parkinson's disease: a systematic review and preliminary meta-analysis.

This systematic review and meta-analysis aimed to identify the determinants for best practice and establish current benchmarks for recovery following reconstructive neurosurgery for people with Parkinson's disease. Eleven studies reporting results for 95 grafted patients were selected on the grounds of using optimal surgical techniques and the Core Assessment Program for Intracerebral Transplantation (CAPIT) protocol for data collection. Consistent trends demonstrating high levels of recovery were identified on most outcome measures. Determinants for best practice were identified as selecting younger patients; using low dose immunosuppression; bilateral grafting; and employing strategies to ensure the quantity and viability of the grafted cells. Secondary analysis of data demonstrated a correlation of rho=0.666 (P<0.05) between increases in striatal dopaminergic activity and UPDRS Motor (off) scores. Overall effect size 'd' was found to be 1.129 UPDRS Motor (off) condition and 0.719 for UPDRS Total (off) condition. The design of the studies and the variable standards for reporting the data precluded the use of more powerful and accurate meta-analyses. It was recommended that the creation of a collaborative database would improve the extraction of data and allow for more powerful statistical analyses for evaluating the overall harm and benefits associated with reconstructive neurosurgery.

A unique cytoplasmic marker for extratesticular Sertoli cells.

In the absence of a definitive cell marker for testis-derived Sertoli cells, their identification in cell culture or in Sertoli cell-facilitated cell transplantation protocols is difficult and limits the creditable evaluation of experimental results. However, the production by prepubertal Sertoli cells of Mullerian inhibiting substance (MIS) presents the possibility of specifically identifying extratesticular Sertoli cells as well as Sertoli cells in situ, by the immunodection of this unique glycoprotein. This study was designed to determine if isolated rat Sertoli cells could be identified by routine immunocytochemistry utilizing an antibody raised against MIS. Sertoli cells immunostained for MIS included Sertoli cells in situ and freshly isolated, cultured and cocultured Sertoli cells, and Sertoli cells structurally integrated with NT2 cells in simulated microgravity. Detection of MIS was also determined by Western blot analysis.