Fernando Carceller

Peripheral nerve regeneration by bone marrow stromal cells

Abstract Adult bone marrow contains stem cells that have attracted interest through their possible use for cell therapy in neurological diseases. Bone marrow stromal cells (MSCs) were harvested from donor adult rats, cultured and pre-labeled with bromodeoxyuridine (BrdU) previously to be injected in the distal stump of transected sciatic nerve of the rats. Distal nerve stump of control rats received culture medium solution. MSCs-treated rats exhibit significant improvement on walking track test at days 18 and 33 compared to controls. Dual immunofluorescence labeling shows that BrdU reactive cells survive in the injected area of transected sciatic nerve at least 33 days after implantation, and almost 5% of BrdU cells express Schwann cell-like phenotype (S100 immunoreactivity). Because MSCs injected in a lesioned peripheral nerve can survive, migrate, differentiate in Schwann cells, and promote functional recovery, they may be an important source for cellular therapy in several neurological diseases.

Glutamate, Glutamine, and GABA as Substrates for the Neuronal and Glial Compartments After Focal Cerebral Ischemia in Rats

BACKGROUND AND PURPOSE Even though the utilization of substrates alternative to glucose may play an important role in the survival of brain cells under ischemic conditions, evidence on changes in substrate selection by the adult brain in vivo during ischemic episodes remains very limited. This study investigates the utilization of glutamate, glutamine, and GABA as fuel by the neuronal and glial tricarboxylic acid cycles of both cerebral hemispheres after partially reversible focal cerebral ischemia (FCI). METHODS Right hemisphere infarct was induced in adult Long-Evans rats by permanent occlusion of the right middle cerebral artery and transitory occlusion of both common carotid arteries. (1,2-13C2) acetate was infused for 60 minutes in the right carotid artery immediately after carotid recirculation had been re-established (1-hour group) or 23 hours later (24-hour group). Extracts from both cerebral hemispheres were prepared and analyzed separately by 13C nuclear magnetic resonance and computer-assisted metabolic modeling. RESULTS FCI decreased the oxidative metabolism of glucose in the brain in a time-dependent manner. Reduced glucose oxidation was compensated for by increased oxidations of (13C) glutamate and (13C) GABA in the astrocytes of the ipsilateral hemispheres of both groups. Increased oxidative metabolism of (13C) glutamine in the neurons was favored by increased activity of the neuronal pyruvate recycling system in the 24-hour group. CONCLUSIONS Data were obtained consistent with time-dependent changes in the utilization of glutamate and GABA or glutamine as metabolic substrates for the glial or neuronal compartments of rat brain after FCI.

Bone marrow stromal cell implantation for peripheral nerve repair.

Abstract Cell therapy using bone marrow stromal cells is a new promising therapy for regenerative medicine. Previous studies demonstrated that local bone marrow stromal cells implantation in the distal stump of transected sciatic nerve of rats promotes early functional recovery. The purpose of this study was to expand on the preliminary research by investigating the long-term efficacy of bone marrow stromal cells using the same experimental setting. Functional test and histological studies demonstrate that bone marrow stromal cell-treated rats exhibit significant improvement on a walking tract test at day 180 after surgery compared with control rats. Taken together, these data suggest that bone marrow stromal cell therapy is a safe and effective strategy for peripheral nerve injuries.

Protection of Rat Myocardium by Mitogenic and Non-Mitogenic Fibroblast Growth Factor During Post-Ischemic Reperfusion

The effects of acidic fibroblast growth factor (FGF-1) and basic fibroblast growth factor (FGF-2) and a non mitogenic form of FGF1 on myocardial ischemia and reperfusion were assessed. Rats underwent 10 minutes of coronary artery occlusion followed by 24 hours of reperfusion. Creatinine kinase content of the affected myocardium showed that both fibroblast growth factors 1 and 2 effectively protected against ischemia reperfusion injury (p < 0.01), and that the vasoactive but nonmitogenic form of the FGF1 was equally protective (p < 0.01 versus control + vehicle). The results were confirmed by light and electron-microscopy histological studies. Histological evaluations after treatment with the non-mitogenic fibroblast growth factor 1 showed that it did not generate the severe hyperplasia and connective tissue disorganization observed with the native mitogenic proteins. The possibility of using a non-mitogenic form of fibroblast growth factor for cardio-protection circumvents many of the potentially undesirable effects that may derive from systemically introducing broad spectrum acting fibroblast growth factors in vivo. This myocardial protection observed 24 hours after the treatment with fibroblast growth factors, and the efficacy of the non-mitogenic form of the protein, also suggest that the protective effect of fibroblast growth factors may be due to the increased blood flow rather than to angiogenesis.

Reduction of Infarct Size by Intra-Arterial Nimodipine Administered at Reperfusion in a Rat Model of Partially Reversible Brain Focal Ischemia

BACKGROUND AND PURPOSE When blood flow to a brain region that has undergone an ischemic attack is reestablished, additional injury is to be expected from the reperfusion. The purpose of the study was to determine the effect of the intra-arterial injection of nimodipine at reperfusion on infarct volume in rats subjected to partially reversible focal neocortical ischemia. METHODS Two groups of Long-Evans rats with transient bilateral common carotid artery occlusion and permanent middle cerebral artery occlusion were subjected to retrograde cannulation of the external carotid artery close to the carotid bifurcation to allow the administration of isotonic saline (group 1) or nimodipine solution (group 2) just before and during reperfusion. The estimate for the actual amount of infarcted cortex was calculated by the volume ratio between the spared cortex in the infarcted hemisphere and the total cortex of the contralateral hemisphere by means of a serological method based on the Cavalieri principle. RESULTS The percentage of cortex that was infarcted in control rats was 63.8 +/- 3.1%, whereas nimodipine-treated rats exhibited a significantly smaller (P < .005) percentage of infarct volume (31.3 +/- 12.7%). CONCLUSIONS Our data show that the intra-arterial injection of nimodipine just before and during reperfusion reduced neocortical infarct volume in rats subjected to partially reversible focal cerebral ischemia.

Morphometric study of focal cerebral ischemia in rats: a stereological evaluation.

An efficient, unbiased stereological method for estimating and evaluating volumetric parameters of cortical infarction is presented. Long-Evans rats were subjected to a standard procedure for achieving focal cerebral ischemia, consisting of permanent ligation of the right middle cerebral artery and temporary occlusion of both common carotid arteries. Animals were killed 24 h later by perfusion fixation and the brains were embedded in celloidin, serially sectioned at 60 microns and stained with Cresyl violet. All cases showed identifiable areas of infarction of variable extent within the territory of the right middle cerebral artery. Involvement of allocortical or subcortical structures was variable and in three cases there were small foci of infarct in the contralateral hemisphere. The absolute volumes of the infarcted tissue and of the spared cortex of both hemispheres were obtained by means of an unbiased estimator of volume based on Cavalieris principle. The best estimate of the actual amount of infarcted cortex was found to be given by the volume ratio between the spared cortex of the right (infarcted) hemisphere and the total cortex of the left (control) hemisphere. This approach avoided the error introduced by the accompanying edema and decreased the observed interanimal variance. Sample size predictions for therapeutic-pharmacological trials show that relatively few animals would be needed using the model of ischemia and quantitative morphometry presented here to detect a 30-40% change in infarct volume.

Intravenous Fibroblast Growth Factor Penetrates the Blood-Brain Barrier and Protects Hippocampal Neurons Against Ischemia-Reperfusion Injury

BACKGROUND Fibroblast growth factors (FGFs) play a role in neuronal survival after brain ischemia when administered intrace-rebrally. However, the clinical problems that chronic intracerebral infusion of FGFs involves may restrict its use. The purpose of this study was to analyze if FGFs administered intravenously might afford neuroprotection against transient brain ischemia in the light of new published data that suggest that these polypeptides cross the blood brain-barrier (BBB). METHODS The efficacy of acidic fibroblast growth factor (aFGF) treatment was analyzed in a gerbil model of 5 min forebrain ischemia followed by 7 days of reperfusion. Native and nonmitogenic aFGF was injected in gerbils as a bolus through a jugular vein at the onset of reperfusion. Control animals received in the same manner vehicle solution alone. Seven days later, neuroprotection was evaluated histologically. Penetration of the FGF across the BBB was assessed by autoradiographic studies in rats. For that purpose, we injected through the jugular vein 0.1 microgram of uniformly labeled native 14C-basic fibroblast growth factor (bFGF), 0.1 microgram of heat-denatured 14C-bFGF, or a coinjection of 14C-bFGF with a 900-fold excess of unlabeled bFGF. Two hours later, animals were killed for morphological studies. RESULTS We report that a venous injection of either native or nonmitogenic form of aFGF after 5 min forebrain ischemia in the gerbil significantly reduced the occurrence of delayed neuronal death (DND) in the CA1 sector of the hippocampus. We also confirmed that blood-borne 14C-bFGF accumulates in CA1 pyramidal neurons.

Single topical application of human recombinant basic fibroblast growth factor (rbfgf) promotes neovascularization in rat cerebral cortex

Basic fibroblast growth factor (bFGF) is a morphogenic, chemotactic, mitogenic, and angiogenic peptide found within the central nervous system (CNS) with potent neurotrophic effects. A potential role in ischemia-induced vascular growth has been suggested for bFGF. In this study, we show that single, topical administration of human recombinant basic fibroblast growth factor (rbFGF) in the rat cerebral cortex promotes capillary overgrowth and might mimic the angiogenic response observed after brain ischemia. The implication of this finding for the therapeutic use of basic FGF in angiogenic therapy is discussed.

Acidic fibroblast growth factor prevents post-axotomy neuronal death of the newborn rat facial nerve

The death of facial motoneurons after axotomy provides a useful tool for studying neurotrophic factors which could prevent motoneuron loss in vivo. The right facial nerve trunk before the postauricular nerve branching of newborn rats was transected at its extracranial exit, and topically treated, at the axotomy site, with either a vehicle solution containing agarose or sucralfate, or acidic fibroblast growth factor (aFGF). Acidic FGF treatment increased the survival of the facial motoneurons from 18% to 70%. These results suggest that aFGF is a neurotrophic factor for motoneurons in vivo and that this growth factor may provide a new basis for the development of treatments to prevent the loss of damaged motoneurons.

Central nervous system distribution of fibroblast growth factor injected into the blood stream

It has been shown that fibroblast growth factors (FGFs) protect hypocampal CAI cells from the effects of transient ischemia when injected either intraventrically or intravenously. The effectiveness of the systemic treatment seems to suggest that FGFs cross the blood-brain barrier to some extent. The appearance of basic fibroblast growth factor (bFGF) in the rat central nervous system after systemic administration have been autoradiographically examined using a 14C uniformly labeled preparation. Our results show that, two hours after a systemic bolus administration, bFGF spots in several populations of neuronal and in non-neural cells except in glial cells. Label accumulation was imperceptible when either 14C-bFGF was heated previously to the systemic injection or was co-administered with an excess of unlabeled bFGF. Our results indicate the existence of a saturable transport system of FGFs across the blood-brain barrier.