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.

Human umbilical cord blood cells directly suppress ischemic oligodendrocyte cell death.

Previous reports have shown that human umbilical cord blood cells (HUCBCs) administered intravenously 48 hr following middle cerebral artery occlusion reduce infarct area and behavioral deficits of rodents. This cellular therapy is potently neuroprotective and antiinflammatory. This study investigates the effect of HUCBC treatment on white matter injury and oligodendrocyte survival in a rat model of ischemia. Intravenous infusion of 10(6) HUCBCs 48 hr poststroke reduced the amount of white matter damage in vivo as seen by quantification of myelin basic protein staining in tissue sections. To determine whether HUCBC treatment was protective via direct effects on oligodendrocytes, cultured oligodendrocytes were studied in an in vitro model of oxygen glucose deprivation. Active caspase 3 immunohistochemistry and the lactate dehydrogenase assay for cytotoxicity were used to determine that HUCBCs provide protection to oligodendrocytes in vitro. Based on these results, it is likely that HUCBC administration directly protects oligodendrocytes and white matter. This effect is likely to contribute to the increased behavioral recovery observed with HUCBC therapy.

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.

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.

Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats.

BACKGROUND AND PURPOSE: Human umbilical cord blood cells (HUCBC) are rich in stem and progenitor cells. In this study we tested whether intravenously infused HUCBC enter brain, survive, differentiate, and improve neurological functional recovery after stroke in rats. In addition, we tested whether ischemic brain tissue extract selectively induces chemotaxis of HUCBC in vitro. METHODS: Adult male Wistar rats were subjected to transient (2-hour) middle cerebral artery occlusion (MCAO). Experimental groups were as follows: group 1, MCAO alone (n=5); group 2, 3x10(6) HUCBC injected into tail vein at 24 hours after MCAO (n=6) (animals of groups 1 and 2 were killed at 14 days after MCAO); group 3, MCAO alone (n=5); group 4, MCAO injected with PBS at 1 day after stroke (n=8); and group 5, 3x10(6) HUCBC injected into tail vein at 7 days after MCAO (n=5). Rats of groups 3, 4, and 5 were killed at 35 days after MCAO. Behavioral tests (rotarod and Modified Neurological Severity Score [mNSS]) were performed. Immunohistochemical staining was used to identify cells derived from HUCBC. Chemotactic activity of ischemia brain tissue extracts toward HUCBC at different time points was evaluated in vitro. RESULTS: Treatment at 24 hours after MCAO with HUCBC significantly improved functional recovery, as evidenced by the rotarod test and mNSS (P<0.05). Treatment at 7 days after MCAO with HUCBC significantly improved function only on the mNSS (P<0.05). Some HUCBC were reactive for the astrocyte marker glial fibrillary acidic protein and the neuronal markers NeuN and microtubule-associated protein 2. In vitro, significant HUCBC migration activity was present at 24 hours after MCAO (P<0.01) compared with normal brain tissue. CONCLUSIONS: Intravenously administered HUCBC enter brain, survive, migrate, and improve functional recovery after stroke. HUCBC transplantation may provide a cell source to treat stroke.

Transplantation of human fetal striatal tissue in Huntington's disease: rationale for clinical studies.

Huntington's disease is a fatal neurological disorder characterized by chorea and deterioration in cognitive and neuropsychiatric function. Primary pathological changes are found in the striatum, where GABAergic neurons undergo degenerative changes. Local interneurons are relatively spared. Here, we describe the rationale for clinical trials of fetal striatal tissue transplantation for the treatment of Huntington's disease. Specifically, the reasons for utilizing tissue derived from the far lateral aspect of the lateral ventricular eminence as a source of striatal tissue will be discussed.

The effects of taurine on hNT neurons transplanted in adult rat striatum.

Taurine acts as an antioxidant able to protect neurons from free radical-mediated cellular damage. Moreover, it modulates the immune response of astrocytes that participate in neurodegenerative processes. The objective of this study was to examine whether taurine can prevent or attenuate the host inflammatory response induced by the xenotransplantation of neurons derived from the human teratocarcinoma cell line (hNT neurons). Male Sprague-Dawley rats were treated IP with either saline or taurine. Animals from both groups were perfused on the 4th or 11th day and the saline or taurine was administered from the start of the study until the day prior to sacrifice. The brains were processed immunohistochemically using antibodies against glial fibrillary acidic protein (GFAP), microglia (OX42), and human nuclear matrix antigen (NuMA). In the saline group, NuMA labeling revealed small grafts on the 4th day and no surviving cells on the 11th day. However, in the group that received taurine there were surviving grafts at both time points. Strong immunoreactivity for GFAP and OX42 was detected in the saline group surrounding the transplant. These effects were reduced in animals receiving taurine. Taken together, these results demonstrated that taurine was able to facilitate graft survival and attenuate the immune response generated by the xenograft.

Trophic effect of porcine Sertoli cells on rat and human ventral mesencephalic cells and hNT neurons in vitro.

The poor survival of embryonic dopaminergic (DA) neurons transplanted into patients with Parkinson's disease (PD) has encouraged researchers to search for new methods to affect the short- as well as long-term survival of these neurons after transplantation. In several previous rodent studies Sertoli cells increased survival of islet cells and chromaffin cells when cotransplanted in vivo. The aims of this study were to investigate whether porcine Sertoli cells had a positive effect on the survival and maturation of rat and human DA neurons, and whether the Sertoli cells had an effect on differentiation of neurons derived from a human teratocarcinoma cell line (hNT neurons). A significant increase of tyrosine hydroxylase (TH)-positive neurons of both rat and human ventral mesencephalic tissue was found when cocultured with Sertoli cells. Furthermore, there was a significantly increased soma size and neurite outgrowth of neurons in the coculture treated group. The Sertoli cell and hNT coculture also revealed an increased number of TH-positive cells. These results demonstrate that the wide variety of proteins and factors secreted by porcine Sertoli cells benefit the survival and maturation of embryonic DA neurons and suggest that cotransplantation of Sertoli cells and embryonic DA neurons may be useful for a cell transplantation therapy in PD.

Capsaicin-resistant vagal afferent fibers in the rat gastrointestinal tract: anatomical identification and functional integrity.

The presence and distribution of vagal fibers and terminals throughout esophagus and gastrointestinal tract that could be anterogradely labeled by nodose ganglion tracer injections was quantitatively assessed in capsaicin- and vehicle-pretreated adult rats, in order to identify the capsaicin-resistant population. Up to 90% of the intraganglionic laminar endings (IGLEs), in the myenteric plexus of the esophagus, and 70-90% in the stomach, as well as 57% of the intramuscular endings or arrays (IMAs) in the fundic stomach survived the capsaicin treatment, while in the upper small intestine only few and in the lower small intestine, the cecum and colon, virtually no IGLEs survived capsaicin treatment. Intramucosal terminals were not assessed. Furthermore, gastric balloon distension-induced c-Fos expression in the dorsal vagal complex was not significantly decreased in capsaicin-treated rats. It is concluded that among primary vagal afferents there is a capsaicin-resistant population that primarily innervates the esophagus and upper gastrointestinal tract, and a capsaicin-sensitive population that innervates mainly the lower tract. At least vagal gastric tension-sensitive afferents also seems to be functionally intact in that they may be capable of synaptically activating second-order neurons in the brainstem.

Sertoli cells enhance the survival of co-transplanted dopamine neurons.

One of the major issues in neural transplantation is the low survival rate (<5%) of transplanted dopamine (DA) neurons [3]. Recently it has been shown that it is possible to enhance the survival of these neurons, which in turn may decrease the amount of tissue that is required for each transplantation patient. The present paper demonstrates a novel approach for enhancing neuronal survival by co-transplantation of neuronal tissue with Testis-derived Sertoli cells (SC). This strategy could improve neuronal survival through the provision of trophic support.

Apoptosis in cultured hNT neurons.

Programmed cell death (apoptosis) is an important mechanism shaping the size of different cell populations within the developing nervous system. In our study we used the NT2/D1 clone originally established from the Ntera 2 cell line to investigate the baseline levels of apoptosis in cultured postmitotic hNT (NT2-N) neurons previously treated for 3, 4 or 5 weeks with retinoic acid (RA) and compared it with apoptosis in NT2 precursors unexposed to RA. First, we examined whether different lengths of exposure to RA might affect baseline apoptotic rate in differentiating hNT neurons. Second, we investigated whether cultured hNT neurons, previously shown to possess dopaminergic characteristics, would be preferentially affected by apoptosis. Using the terminal deoxynucleotidyl transferase (tdt)-labeling technique we found that the postmitotic hNT neuronal cells exposed to RA demonstrated significantly higher numbers of apoptotic cells (12.5-15.8%) in comparison to rapidly dividing NT2 precursor cell line (3.6-4.4%) at both studied (1 and 5 days in vitro, DIV) time points. Similar apoptotic nuclear morphology, including a variable extent of nuclear fragmentation was observed in all examined hNT cultures. On the other hand, the incidence of apoptotic nuclei was rare in cultures of NT2 precursors not subjected to RA treatment. Combined immunocytochemistry for tyrosine hydroxylase (TH) and Hoechst staining revealed dopaminergic hNT neurons destined to die. Our double-labeling studies have demonstrated that only a subset of TH-positive hNT cells had condensed chromatin after 1 (approx. 15%) and 5 (approx. 20%) DIV. NT2 precursors were not TH-positive. Collectively, our results demonstrated that exposure to differentiating agent RA triggers an apoptotic commitment in a subset of postmitotic hNT neurons. These results suggest that this cell line may serve as a model of neuronal development to test various pathogenic factors implicated in the etiology of Parkinson's disease (PD), as well as to screen numerous pharmacological treatments that may slow or prevent dopaminergic deterioration.

Dopaminergic phenotype of hNT cells in vitro.

We investigated the catecholaminergic nature of cultured hNT neurons previously treated either for 4 or 5 weeks with retinoic acid (RA). There were significantly more tyrosine hydroxylase (TH)-positive neurons (60%) in cultures treated for 4 weeks with RA compared to 5 week-treated cultures (

Preparation of cell suspensions for co-transplantation: methodological considerations.

The purpose of the current study was to determine the optimal strategy for preparing cell suspensions for co-transplantation. In the first experiment, the number of Sertoli cell (SC) aggregates and the number of tyrosine hydroxylase positive neurons were compared over time when cell suspensions of Sertoli or ventral mesencephalic cells were kept as a co-suspension mixed at 0 h. Cells from each suspension were dispensed onto glass slides in a manner similar to transplantation. When dispensed in this manner, the number of SC aggregates and TH-positive neurons decreased over 4 h. In experiment 2, the cell suspensions were mixed just prior to injection at each of four timepoints, the number of aggregates and TH neurons was consistent over time. Clearly this latter strategy resulted consistent recovery of both cell types for transplants up to 3 h after suspension preparation.

Expression of fos-related antigen-2 in rat hippocampus after middle cerebral arterial occlusion.

AP-1 transcription factors have been shown to be induced in the brain after ischemic injury. However, their roles in neuronal survival or death have yet to be defined. Here, we report the discovery of elevated nuclear levels of fos-related antigen-2 (FRA-2) in the nuclei of hippocampal neurons seven days after middle cerebral artery occlusion (MCAO). Expression of FRA-2 and AP-1 DNA binding activity is elevated in hippocampi ipsilateral as well as contralateral to MCAO. Using Fluoro-Jade staining as a marker of neurodegeneration, FRA-2 was not found to be expressed in degenerating neurons. Thus, FRA-2 is expressed in neurons that survive ischemic insult suggesting a role for this transcription factor in neuronal adaptation to the post-injury state.

Does the integrated level of all plasma nutrients control daily food intake?

Studies on two different types of one-way crossed-intestines rats have shown that daily food intake is controlled by either endogenous gut signals or absorbed nutrients and their metabolic consequences, or both. If the amount of incoming ingested food is metered somewhere in the body, this could only occur in the gut or liver. The capacity of the liver to determine the amount of water-soluble nutrient absorbed was assessed by portacaval shunt and found to be inadequate. Infusion of nutrients directly into the bloodstream show that plasma nutrients provide part of the signal that inhibits daily food intake, but that endogenous gut signals must play some role. Insulin, an important hormone in the movement of plasma nutrients into cells, was found to stimulate food intake at low infusion doses. IV nutrients raise the level of plasma nutrients and lower daily food intake, while insulin, which inhibits the release of endogenous fuels and moves exogenous fuels into cells, lowers plasma nutrients and stimulates daily intake. Thus, the integrated level of all plasma nutrients may be a major controller of daily food intake.

hNT neurons delay onset of motor deficits in a model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease that manifests as a progressive muscular weakness leading to paralysis and death. Because of the diffuse nature of the motor neuron death, this disease is not considered a good candidate for treatment through neural transplantation. The purpose of this study was to show that transplantation of human neuron-like cells (hNT neurons) into the spinal cord of a transgenic ALS mouse model would improve motor deficits. The hNT neurons were transplanted bilaterally into L4-L5 spinal cord of the transgenic mice ( approximately 8 weeks of age), and the animals were evaluated on health and behavioral measures. The animals were perfused, and immunohistochemistry was performed to identify the transplanted cells. Transplantation of the hNT neurons into the spinal cord delayed the onset of motor behavioral symptoms. This was the first demonstration that even localized transplantation of neural cells directly into the parenchyma could improve motor function in an ALS model. Further study is needed to delineate the mechanism underlying these effects. This therapeutic approach has the potential to restore neural transmission, thereby improving quality of life for the ALS patient and possibly extend life expectancy.

In vitro induction and in vivo expression of bcl-2 in the hNT neurons.

Bcl-2 encodes membrane-associated proteins that suppress programmed cell death in cells of various origins. Compelling evidence suggests that bcl-2 is also involved in neuronal differentiation and axonal regeneration. The human Neuro-Teratocarcinoma (hNT) neurons constitute a terminally differentiated human neuronal cell line that is derived from the Ntera-2/clone D1 (NT2) precursors upon retinoic acid (RA) treatment. After transplantation into the central nervous system (CNS), the hNT neurons survive, engraft, maintain their neuronal identity, and extend long neurite outgrowth. We were particularly interested in the intracellular determinants that confer these post-transplant characteristics to the hNT neurons. Thus, we asked whether the hNT neurons express bcl-2 after transplantation into the rat striatum and if RA induction of the neuronal lineage is mediated by bcl-2. The grafted hNT neurons were first identified using three different antibodies that recognize human-specific epitopes, anti-hMit, anti-hNuc, and NuMA. After a 1-month post-transplant survival time, NuMA immunostaining revealed that 12% of the hNT neurons survived the transplantation. These neurons extended long neuritic processes within the striatum, as demonstrated using the human-specific antibody against the midsize neurofilament subunit HO14. Importantly, we found that 85% of the implanted hNT neurons expressed bcl-2 and that the in vitro induction of the neuronal lineage from the NT2 precursors with RA resulted in an upregulation of bcl-2 expression. Together, these data suggest that the differentiation of the hNT neurons to a neuronal lineage could be mediated at least partially by bcl-2.

Induction of presenilins in the rat brain after middle cerebral arterial occlusion.

In the present study, we have examined the expression of both presenilins in the rat hippocampus, cortex, striatum, and cerebellum after middle cerebral artery occlusion (MCA-O), an animal model of ischemia. The cortex showed the greatest increase in PS mRNA levels (7-10-fold) at 4 and 8 days posttreatment. Presenilin-1 (PS-1) levels in the contralateral cortex were significantly increased 1 day after MCA-O. In comparison, PS mRNA content was only modestly elevated in the hippocampus and striatum at 4 and 8 days after MCA-O (30-100% changes). Other Alzheimer's disease (AD)-related genes, amyloid precursor protein and apolipoprotein E, are induced in brain injury suggesting that these AD-related genes may well be components of a brain-injury response. Thus, a breakdown in this response via cerebrovascular disease and/or genetic mutation may contribute to AD pathology.

In vitro and in vivo characterization of hNT neuron neurotransmitter phenotypes.

The hNT neuron exhibits many characteristics of neuroepithelial precursor cells, making them an excellent model to study neuronal plasticity in vitro and in vivo. These cells express a number of neurotransmitters in vitro, including dopamine, gamma-aminobutyric acid and acetylcholine. However, there have been few reports of the neurotransmitters that hNT neurons express in vivo. The present study examined whether hNT neurons express the same neurotransmitters in vivo as they do in vitro. First, the expression of tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD), choline acetyltransferase (ChAT) and the human specific nuclear marker NuMA by hNT neurons was confirmed. Nineteen normal animals were then transplanted with 80,000 hNT neurons aimed at the striatum, hippocampus or cerebral cortex. Five additional animals received injections of medium. All animals received daily intraperitoneal injections of cyclosporine (10 mg/kg) and survived 30 days. Sections through the transplants were examined for NuMA-positive hNT neurons, and for the presence of the three neurotransmitter markers: TH, GAD and ChAT. The hNT neurons were found in the striatum and cortex. Of the hNT neurons found within the rat striatum, 33% were ChAT-positive. In the cortex, only 4% of the neurons expressed ChAT. No GAD-positive hNT neurons were detected at either site. No NuMA-positive neurons were found in the hippocampus. The implanted hNT neurons did not induce activation of astrocytes as determined by immunocytochemistry for glial fibrillary acidic protein (GFAP). Moreover, no hNT neuron was found to express GFAP in vivo. Together, these data suggest that the hNT neurons engraft in the new host tissue, maintain their neuronal identity and may be guided in differentiation according to local environmental cues.