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Sertoli cells (SCs) are testis-derived cells that secrete trophic factors important for the development of germ cells. Both porcine and rat SCs have been used as graft facilitators - neonatal porcine SCs to support islets in diabetes and 15-day-old rat SCs to enhance dopaminergic neuron transplants in Parkinson's disease models. However, there has never been a study examining the optimal SCs preparation to enhance tyrosine hydroxylase expression in the ventral mesencephalon (VM) neuron. The aim of this study was to compare the ability of both rat and porcine SCs to enhance tyrosine hydroxylase expression (TH) and neuronal survival at the same postnatal developmental ages. The SCs were isolated from 1-, 9-, or 15-day-old rat, or neonate (2-5 days), 2-month, or 4-month-old pig, and co-cultured with VM tissue from 13.5-day-old embryos. Our results showed that VM neurons co-cultured with SCs dispersed over the culture plate and had extensive neuritic outgrowth, while VM neurons cultured alone tended to cluster together forming a mass of cells with limited neurite outgrowth. TH expression was significantly increased when VM neurons were co-cultured with 15-day rat SCs or 2-month pig SCs but not when the cells were co-cultured with other ages of SCs. This suggests that secretion of trophic factors by SCs varies according to the developmental age, and it is critical for the success of graft facilitation that SCs from the appropriate age and species be used.

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.

Nitric oxide (NO) is a gaseous intercellular messenger produced by the enzyme nitric oxide synthase (NOS). In this study, we used two different techniques-nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and NOS immunocytochemistry-to demonstrate that NOS is present in a pair of identifiable cells in the central nervous system of the nudibranch Melibe leonina. In the Melibe brain, NADPH-d histochemistry revealed only a single pair of bilaterally symmetrical cells in the cerebropleural ganglia. NOS activity also was found in the neuropil of the cerebral, pedal, and buccal ganglia; in the tentacles of the oral hood; in the sensory end of the rhinophores; and in the epithelial tissue of the mouth, preputium, and glans penis. Immunocytochemistry using NOS antisera corroborated the results of the NADPH-d histochemistry by staining the same two cells in the cerebropleural ganglia. Each of these identifiable nitrergic neurons projects into the ipsilateral pedal ganglion. Because the pedal ganglia play a critical role in the control of locomotion, our results provide morphological evidence suggesting that NO may influence swimming or crawling in Melibe leonina., (Copyright 2001 Wiley-Liss, Inc.)

This study investigated the temporal expression and cell subtype distribution of activated caspase-3 following cortical impact-induced traumatic brain injury in rats. The animals were killed and examined for protein expression of the proteolytically active subunit of caspase-3, p18, at intervals from 6 h to 14 days after injury. In addition, we also investigated the effect of caspase-3 activation on proteolysis of the cytoskeletal protein alpha-spectrin. Increased protein levels of p18 and the caspase-3-specific 120-kDa breakdown product to alpha-spectrin were seen in the cortex ipsilateral to the injury site from 6 to 72 h after the trauma. Immunohistological examinations revealed increased expression of p18 in neurons, astrocytes, and oligodendrocytes from 6 to 72 h following impact injury. In contrast, no evidence of caspase-3 activation was seen in microglia at all time points investigated. Quantitative analysis of caspase-3-positive cells revealed that the number of caspase-3-positive neurons exceeded the number of caspase-3-positive glia cells from 6 to 72 h after injury. Moreover, concurrent assessment of nuclear histopathology using hematoxylin identified p18-immunopositive cells exhibiting apoptotic-like morphological profiles in the cortex ipsilateral to the injury site. In contrast, no evidence of increased p18 expression or alpha-spectrin proteolysis was seen in the ipsilateral hippocampus, contralateral cortex, or hippocampus up to 14 days after the impact. Our results are the first to demonstrate the concurrent expression of activated caspase-3 in different CNS cells after traumatic brain injury in the rat. Our findings also suggest a contributory role of activated caspase-3 in neuronal and glial apoptotic degeneration after experimental TBI in vivo.

Traumatic brain injury (TBI) results in numerous central and systemic responses that complicate interpretation of the effects of the primary mechanical trauma. For this reason, several in vitro models of mechanical cell injury have recently been developed that allow more precise control over intra- and extracellular environments than is possible in vivo. Although we recently reported that calpain and caspase-3 proteases are activated after TBI in rats, the role of calpain and/or caspase-3 has not been examined in any in vitro model of mechanical cell injury. In this investigation, varying magnitudes of rapid mechanical cell stretch were used to examine processing of the cytoskeletal protein alpha-spectrin (280 kDa) to a signature 145-kDa fragment by calpain and to the apoptotic-linked 120-kDa fragment by caspase-3 in septo-hippocampal cell cultures. Additionally, effects of stretch injury on cell viability and morphology were assayed. One hour after injury, maximal release of cytosolic lactate dehydrogenase and nuclear propidium iodide uptake were associated with peak accumulations of the calpain-specific 145-kDa fragment to alpha-spectrin at each injury level. The acute period of calpain activation (1-6 h) was associated with subpopulations of nuclear morphological alterations that appeared necrotic (hyperchromatism) or apoptotic (condensed, shrunken nuclei). In contrast, caspase-3 processing of alpha-spectrin to the apoptotic-linked 120-kDa fragment was only detected 24 h after moderate, but not mild or severe injury. The period of caspase-3 activation was predominantly associated with nuclear shrinkage, fragmentation, and apoptotic body formation characteristic of apoptosis. Results of this study indicate that rapid mechanical stretch injury to septo-hippocampal cell cultures replicates several important biochemical and morphological alterations commonly observed in vivo brain injury, although important differences were also noted.

Apoptotic cell death has been observed in both neurodegenerative diseases and acute neurological traumas such as ischemia, spinal cord injury, and traumatic brain injury (TBI). Recent studies employing different models of TBI have described morphological and biochemical changes characteristic of apoptosis following injury. However, no study has examined the temporal profile of apoptosis following controlled cortical impact (CCI) injury in the rat. In addition, the relative frequency of apoptotic profiles in different cell types (neurons versus glia) following CCI has yet to be investigated. In the present experiments, injured cortex was subjected to DNA electrophoresis, and serial sections from the contusion area were stained with hematoxylin and eosin or Hoechst 33258 or double-labeled with TUNEL and neuronal or glial markers. The results of the present study indicate that CCI produces a substantial amount of DNA damage associated with both apoptotic-like and necrotic-like cell death phenotypes primarily at the site of cortical impact and focal contusion. DNA damage, as measured by TUNEL and DNA electrophoresis, was most apparent 1 day following injury and absent by 14 days post-TBI. However, quantitative analysis showed that the majority of TUNEL-positive cells failed to exhibit apoptotic-like morphology and were probably undergoing necrosis. In addition, apoptotic-like morphology was predominantly observed in neurons compared to astrocytes. The present study provides further evidence that apoptosis is involved in the pathology of TBI and could contribute to some of the ensuing cell death following injury.