Transplantation of Human Neural Progenitor Cells (NPC) into Putamina of Parkinsonian Patients: A Case Series Study, Safety and Efficacy Four Years after Surgery.

Individuals with Parkinson's disease (PD) suffer from motor and mental disturbances due to degeneration of dopaminergic and non-dopaminergic neuronal systems. Although they provide temporary symptom relief, current treatments fail to control motor and non-motor alterations or to arrest disease progression. Aiming to explore safety and possible motor and neuropsychological benefits of a novel strategy to improve the PD condition, a case series study was designed for brain grafting of human neural progenitor cells (NPCs) to a group of eight patients with moderate PD. A NPC line, expressing Oct-4 and Sox-2, was manufactured and characterized. Using stereotactic surgery, NPC suspensions were bilaterally injected into patients' dorsal putamina. Cyclosporine A was given for 10 days prior to surgery and continued for 1 month thereafter. Neurological, neuropsychological, and brain imaging evaluations were performed pre-operatively, 1, 2, and 4 years post-surgery. Seven of eight patients have completed 4-year follow-up. The procedure proved to be safe, with no immune responses against the transplant, and no adverse effects. One year after cell grafting, all but one of the seven patients completing the study showed various degrees of motor improvement, and five of them showed better response to medication. PET imaging showed a trend toward enhanced midbrain dopaminergic activity. By their 4-year evaluation, improvements somewhat decreased but remained better than at baseline. Neuropsychological changes were minor, if at all. The intervention appears to be safe. At 4 years post-transplantation we report that undifferentiated NPCs can be delivered safely by stereotaxis to both putamina of patients with PD without causing adverse effects. In 6/7 patients in OFF condition improvement in UPDRS III was observed. PET functional scans suggest enhanced putaminal dopaminergic neurotransmission that could correlate with improved motor function, and better response to L-DOPA. Patients' neuropsychological scores were unaffected by grafting. Trial Registration: Fetal derived stem cells for Parkinson's disease https://doi.org/10.1186/ISRCTN39104513Reg#ISRCTN39104513.

Characterization of spinal subarachnoid bleeding associated to graded traumatic spinal cord injury in the rat.

STUDY DESIGN: Observational study in rats subjected to traumatic spinal cord injury (SCI). OBJECTIVES: To describe the features of spinal subarachnoid bleeding (SSB) occurring after graded SCI. SSB after SCI has been reported previously, but has not been studied systematically despite the fact that cerebral subarachnoid bleeding often produces severe neurological damage. SETTING: Mexico. METHODS: Anesthetized rats were subjected to mild or severe spinal cord contusion at T9. Occurrence, size, progression and location of SSB were characterized morphologically and scored from T7-T12 at 1 h and 1, 3 and 7 days post injury. Besides, contusions were videotaped to visualize bleeding at the moment of impact. RESULTS: SSB started immediately after contusion (severe or mild) and decreased gradually over time. For all vertebral segments, at all time points examined by histology, 48% of areas scored after severe contusion showed bleeding: 25% minor, 17% moderate and 6% major. After mild contusion, only 15% showed bleeding: 13 minor and 2% moderate. Maximum bleeding occurred early after injury in dorsal area of the epicenter in 100% of severe contusions (6% minor, 38 moderate and 56% major), and in 69% of mild contusions (63 minor and 6% moderate). CONCLUSION: Here, we detail SSB patterns occurring after graded SCI. Further studies are warranted to elucidate the possible role extramedullary events, such as SSB, in the pathophysiology of SCI that might encourage the development of new strategies for its management.

Changes in renal function during acute spinal cord injury: implications for pharmacotherapy.

STUDY DESIGN: Laboratory investigation in rats submitted to experimental spinal cord injury (SCI). OBJECTIVE: To characterize changes in renal function during acute SCI. METHODS: Sprague Dawley rats were subjected to severe spinal cord contusion at T8 level or to laminectomy as control. Twenty-four hours after spine surgery, clearance assessments of a single dose of iohexol (120 mg kg(-1)) or of p-aminohippuric acid (PAH, 100 mg kg(-1)) were used to evaluate glomerular filtration rate (GFR) and tubular secretion (TS), respectively. Blood sampling was used to determine concentrations of both compounds by high-performance liquid chromatography for pharmacokinetic measurements. RESULTS: Iohexol clearance decreased significantly after injury, which resulted in increased concentrations and half-life of iohexol in blood; PAH clearance remained unchanged. CONCLUSION: GFR but not TS is altered during spinal shock. These observations should be of interest to professionals caring for early cord-injured patients, in order to prevent toxicity and therapeutic failure when administering drugs eliminated by the kidney.

Contrasting effects of cord injury on intravenous and oral pharmacokinetics of diclofenac: a drug with intermediate hepatic extraction.

STUDY DESIGN: Laboratory investigation in rats submitted to experimental spinal cord injury (SCI). OBJECTIVE: To determine the effect of acute SCI on the pharmacokinetics of diclofenac, a marker drug of intermediate hepatic extraction, administered by the intravenous and the oral routes. METHODS: Female Wistar rats were submitted to complete section of the spinal cord at the T8 level. SCI and sham-injured rats received 3.2 mg kg(-1) of diclofenac sodium either intravenously or orally, diclofenac concentration was measured in whole blood samples and pharmacokinetic parameters were estimated. Diclofenac was not selected as test drug because of its therapeutic properties, but because to its biopharmaceutical properties, that is, intermediate hepatic extraction. RESULTS: Diclofenac bioavailability after intravenous administration was increased in injured rats compared with controls due to a reduced clearance. In contrast, oral diclofenac bioavailability was diminished in SCI animals due to a reduction in drug absorption, which overrides the effect on clearance. CONCLUSION: Acute SCI induces significant pharmacokinetic changes for diclofenac, a marker drug with intermediate hepatic extraction. SCI-induced pharmacokinetic changes are not only determined by injury characteristics, but also by the route of administration and the biopharmaceutical properties of the studied drug.

Lack of neuroprotection with pharmacological pretreatment in a paradigm for anticipated spinal cord lesions.

BACKGROUND: In humans elective spine surgery can cause iatrogenic spinal cord injury (SCI). Efforts for neuroprotection have been directed to avoid mechanical injury by using intraoperative monitoring and improving surgical techniques. There is, however, uncertainty regarding the efficacy of neuroprotective drugs. STUDY DESIGN: Experimental study on the effectiveness of pharmacological neuroprotection in an animal model of spine surgery simulating anticipated mechanically induced neurological damage. OBJECTIVE: To compare the efficacy of four drugs to protect against the neurological effects of iatrogenic SCI. SETTING: Research Unit for Neurological Diseases, IMSS-Proyecto Camina, Mexico City, Mexico. METHODS: Erythropoietin, melatonin, cyclosporine-A and methylprednisolone were administered to rats before, during and after controlled spinal cord contusion of mild intensity. Dosage was in accordance with their pharmacokinetic properties and experience gained with experimental SCI. Drug efficacy was assessed by motor function recovery over a period of 6 weeks and by spinal cord morphometry. RESULTS: Compared with animals treated with saline, the drug-treated groups showed no differences in their locomotor performance, nor in the amount of spared cord tissue. Notably, spontaneous activity was significantly reduced in rats treated with cyclosporine-A. CONCLUSION: The neuroprotectant drugs used here perioperatively did not reduce the extent of neurological damage in a model simulating iatrogenic SCI. Therefore, for now, the only protection in elective spine surgery is avoidance of primary injury altogether.

Glutathione monoethyl ester improves functional recovery, enhances neuron survival, and stabilizes spinal cord blood flow after spinal cord injury in rats.

Secondary damage after spinal cord (SC) injury remains without a clinically effective drug treatment. To explore the neuroprotective effects of cell-permeable reduced glutathione monoethyl ester (GSHE), rats subjected to SC contusion using the New York University impactor were randomly assigned to receive intraperitoneally GSHE (total dose of 12 mg/kg), methylprednisolone sodium succinate (total dose of 120 mg/kg), or saline solution as vehicle. Motor function, assessed using the Basso-Beattie-Bresnahan scale for 8 weeks, was significantly better in GSHE (11.2+/-0.6, mean+/-S.E.M., n=8, at 8 weeks) than methylprednisolone (9.3+/-0.6) and vehicle (9.4+/-0.7) groups. The number of neurons in the red nuclei labeled with FluoroRuby placed caudally to the injury site was significantly higher in GSHE (158+/-9.3 mean+/-S.E.M., n=4) compared with methylprednisolone (53+/-14.7) and vehicle (46+/-16.4) groups. Differences in the amount of spared SC tissue at the epicenter and neighboring areas were not significant among experimental groups. In a second series of experiments, using similar treatment groups (n=6), regional changes in microvascular SC blood flow were evaluated for 100 min by laser-Doppler flowmetry after clip compression injury. SC blood flow fell in vehicle-treated rats 20% below baseline and increased significantly with methylprednisolone approximately 12% above baseline; changes were not greater than 5% in rats given GSHE. In conclusion, GSHE given to rats early after moderate SC contusion/compression improves functional outcome and red nuclei neuron survival significantly better than methylprednisolone and vehicle, and stabilizes SC blood flow. These results support further investigation of reduced glutathione supplementation after acute SC injury for future clinical application.

Transitory expression of NADPH diaphorase (NOS) in axonal swellings after spinal cord injury.

To investigate the sites of nitric oxide synthase (NOS) expression after a spinal cord (SC) injury, NADPH-d diaphorase histochemistry was performed in the SC of adult rats sacrificed at different times from 1 h to 90 days after both SC contusion or transection. NOS could first be seen 12 h after injury in axonal swellings (AS) (club shaped structures at the tip of damage axons, associated with tissue destruction). NOS expression reached a maximum 3 days after injury, and gradually disappeared after 7 days. Finally, AS collapsed leaving behind microcysts. NOS expression and the consequent production of nitric oxide could be involved in the pathophysiology of the secondary damage, and/or could reflect a failed attempt for axonal regeneration.

Vasopressinergic, oxytocinergic, and somatostatinergic neuronal activity after adrenalectomy and immobilization stress.

Twenty days after bilateral adrenalectomy (ADX) or immediately after the last of three 6-h long immobilization periods, the levels of hypothalamic and neurohypophyseal L-[35S]Cys-labeled arginine vasopressin (AVP), oxytocin (OT), and somatostatin-14 (SRIF) (only stressed animals) were measured simultaneously in male Wistar rats, after third ventricular administration of the labeled precursor, via guide-cannulae. The acetic acid-extracted labeled peptide fractions were purified by two sequential HPLC steps. After a 4 h period of labeling, only L-[35S]Cys-AVP was selectively increased in the hypothalami of ADX-ized rats, compared to the sham-operated animals, possibly reflecting a significant activation of the paraventricular parvocellular (PVC) AVP/corticotropin-releasing factor (CRF) neurons. The increased accumulation of neurohypophyseal L-[35S]Cys-labeled AVP and OT in these animals, without changes in the endogenous levels of these peptides, as measured by UV absorbance, also suggests a moderate activation of the magnocellular (MGC) AVP and OT neurons, as a consequence of adrenal insufficiency. In response to immobilization stress, levels of L-[35S]Cys-OT were selectively increased in the hypothalami and corresponding neurohypophyses, 2 h and 4 h after receiving the label, concomitantly with a statistically significant reduction in the stores of OT in the neural lobes. AVP and SRIF biosynthesis remained unaffected by immobilization; the neurohypophyseal AVP stores likewise remained unchanged. These observations suggest the selective activation of MGC-OT neurons in response to chronic immobilization stress. Selective increases in hypothalamic L-[35S]Cys-AVP in ADX-ized rats, and in hypothalamic L-[35S]Cys-OT in chronically stress-immobilized rats, are presented as a measure of PVC-AVP/CRF and MGC-OT neuronal activation, respectively.

Vitreous body glutamate concentration in dogs with glaucoma.

OBJECTIVE: To analyze the vitreal amino acid concentrations in dogs with breed-related primary glaucoma to determine whether excitotoxic amino acids associated with retinal genglion cell death in other species were present in affected dogs. SAMPLES: 11 normal control and 10 glaucomatous canine eyes. PROCEDURE: Amino acid analyses were performed by high-pressure liquid chromatography in masked manner. RESULTS: Eyes from dogs with primary glaucoma had significantly high vitreal glutamate concentration, compared with values for eyes of clinically normal control dogs. Mean (+/-SD) glutamate concentrations were 31.7 +/- 12.4 and 6.9 +/- 6.3 microM in glaucomatous and normal eyes, respectively (P < 0.0001). Eyes from dogs with glaucoma also had lower vitreal glycine (37.0 +/- 17.0 vs 59.4 +/- 28.2 microM; P < 0.043) and higher of vitreal tryptophan (39.0 +/- 22.8 vs 17.5 +/- 11.2 microM; P < 0.012) concentrations, compared with values for normal eyes. CONCLUSION: Glutamate concentration potentially toxic to retinal ganglion cells is associated with the pathogenesis of primary glaucoma in dogs. Increased glutamate concentration provides evidence of an ischemic mechanism for retinal ganglion cell death and optic nerve atrophy in dogs with glaucoma. CLINICAL RELEVANCE: The emphasis on reduction and normalization of high intraocular pressure as the primary focus of treatment for glaucoma in dogs should be augmented by other therapeutic approaches.

Use of cyclosporin-A in experimental spinal cord injury: design of a dosing strategy to maintain therapeutic levels.

Cyclosporin-A (CsA) is frequently used as an immunosuppressive agent in experimental transplantations. CsA has been used in nervous tissue transplants in spinal cord injury (SCI). However, optimal results have not been obtained. This is likely due to the fact that SCI alters CsA pharmacokinetics and hence fixed dose regimens are not adequate. In this study, several CsA dosing regimens were evaluated in Long-Evans female rats subjected to a severe low thoracic (T8) SCI by the contusion method. Serum CsA concentrations were measured to determine which dosing regimen allowed CsA levels to be maintained within the therapeutic window. It was found that administration of 2.5 mg/kg/12 h intraperitoneally during the first 2 days after SCI (acute phase) followed by 5 mg/kg/12 h orally thereafter (subacute and chronic phases) yields CsA circulating levels within the therapeutic window, i.e., 0.120-0.275 microgram/mL. This dosing regimen represents a suitable alternative to fixed dosing to achieve an optimal CsA-induced immunosuppression in experimental models of SCI.

Alteration of cyclosporin-A pharmacokinetics after experimental spinal cord injury.

The pharmacokinetics of the immunosuppressive agent cyclosporin-A (CsA) were studied in rats submitted to spinal cord (SC) injury. A single CsA 10 mg/kg dose was given either intraperitoneally (i.p.) or orally to rats submitted to experimental SC injury at the T8 level. Twenty four hours after lesion (acute stage of SC injury) i.p. CsA bioavailability was increased, while t1/2 was prolonged. However, oral bioavailability was reduced. Seven weeks after lesion (chronic stage of SC injury) CsA bioavailability, by either route, was not significantly different from control values. Results indicate that parenteral CsA bioavailability is increased during the acute stage of SC lesion, probably due to an impaired elimination. Oral bioavailability, however, is decreased, since there is also an important reduction in gastrointestinal CsA absorption that overrides the effect of impaired elimination. Alterations in CsA pharmacokinetics appear to revert during the chronic stage of SC injury. Changes in CsA bioavailability, depending on the route of administration and on time, must be considered to design an adequate immunosuppressive treatment in SC injury.

Histochemical evidence of the increased expression of nicotinamide adenine dinucleotide phosphate-dependent diaphorase in neurons of the myenteric plexus after acute spinal cord injury in adult rats.

The expression of nitric oxide synthase in neurons of the gastrointestinal tract (GIT) after experimental spinal cord injury (SCI) was assessed in adult rats contused at T8. One day and 10 weeks after injury, specimens along the GIT were studied for NADPH-diaphorase histochemistry. A significant increase in the number of positive cell bodies and fibers in the myenteric plexus were observed 1 day after SCI, as compared to specimens from control and chronically injured rats, with the exception of the colon, which showed unchanged or decreased number of positive neurons in the acute and chronic stages, respectively. Positive neurons in the submucous plexus remained unchanged, excepting an increase in the colon after acute SCI, and a decrease in the duodenum in chronically injured rats. The altered nitric oxide neurotransmission in the GIT may be relevant to its reduced motility after SCI.

Fetal striatal homotransplantation for Huntington's disease: first two case reports.

Based on the successful use of fetal striatal brain grafting in the restoration of striatal function in rat and nonhuman primate models of Huntington's disease, as well as on the evidence for the clinical potential of fetal brain grafting in the treatment of Parkinson's disease, homotopic fetal striatal homotransplantations were performed in two huntingtonians. Case 1 was a 37 year-old female with moderate to severe Huntington's disease of 9 years evolution; case 2 was a 29 year-old male with mild Huntington's disease of 5 years evolution. Using open microsurgery, each patient was implanted to the ventricular wall of the right caudate nucleus with both striata from a 13 week-old and a 12 week-old human fetus, respectively. Since surgery both patients were kept on cyclosporine A. Surgery produced no damaging effect to either patient. The time course of the neurological progression of their disease, spanning 33 months for case 1, and 16 months for case 2, reveal that the disease in both patients has progressed more slowly in relation to their preoperative state. Although presently it is not possible to determine to what extent, surgery has modified the course of their disease, or if it will continue to have an effect on it, these surgeries represent the first step towards the development of brain grafting for Huntington's disease.

Magnetic resonance imaging of the normal and chronically injured adult rat spinal cord in vivo.

We assessed the capacity of MRI to show and characterise the spinal cord (SC) in vivo in normal and chronically injured adult rats. In the chronically injured animals the SC was studied by MRI and histological examination. MRI was performed at 1.5 T, using gradient-echo and spin-echo (SE) sequences, the latter with and without gadolinium-DTPA (Gd-DTPA). Several positions were tried for good alignment and to diminish interference by respiratory movements. Images of the SC were obtained in sagittal, coronal, and axial planes. Normal SC was observed as a continuous intensity in both sequences, although contrast resolution was better using SE; it was not possible to differentiate the grey and white matter. Low signal was seen in the damaged area in chronically injured rats, which corresponded to cysts, trabeculae, mononuclear infiltrate, and fibroglial wall on histological examination. Gd-DTPA failed to enhance the SC in normal or chronically injured rats. It did, however, cause enhancement of the lesion after acute SC injury.

Development of post-traumatic cysts in the spinal cord of rats-subjected to severe spinal cord contusion.

To study the development of post-traumatic spinal cord (SC) cysts, and their fine anatomic characteristics, rats were subjected to severe SC contusion. Specimens were analyzed from day 1 to 1 year post-injury. Using conventional light, and transmission and scanning electron microscopy, three stages were typified, namely: necrosis, repair, and stability. The final cell composition and thickness of the cyst walls were not uniform. Astrocytes, fibroblasts, ependymal cells, and collagen fibers were the main constituents. Chronic inflammatory cells were also observed. The neuropathologic characterization of posttraumatic SC cysts could be useful in planning strategies for SC reconstruction at different times post-injury.

Dopamine beta-hydroxylase immunoreactivity in human cerebrospinal fluid: properties, relationship to central noradrenergic neuronal activity and variation in Parkinson's disease and congenital dopamine beta-hydroxylase deficiency.

1. Dopamine beta-hydroxylase is stored and released with catecholamines by exocytosis from secretory vesicles in noradrenergic neurons and chromaffin cells. Although dopamine beta-hydroxylase enzymic activity is measurable in cerebrospinal fluid, such activity is unstable, and its relationship to central noradrenergic neuronal activity in humans is not clearly established. To explore the significance of cerebrospinal fluid dopamine beta-hydroxylase, we applied a homologous human dopamine beta-hydroxylase radioimmunoassay to cerebrospinal fluid, in order to characterize the properties and stability of cerebrospinal fluid dopamine beta-hydroxylase, as well as its relationship to central noradrenergic neuronal activity and its variation in disease states such as hypertension, renal failure, Parkinsonism and congenital dopamine beta-hydroxylase deficiency. 2. Authentic, physically stable dopamine beta-hydroxylase immunoreactivity was present in normal human cerebrospinal fluid at a concentration of 31.3 +/- 1.4 ng/ml (range: 18.5-52.5 ng/ml), but at a 283 +/- 27-fold lower concentration than that found in plasma. Cerebrospinal fluid and plasma dopamine beta-hydroxylase concentrations were correlated (r = 0.67, P = 0.001). Some degree of local central nervous system control of cerebrospinal fluid dopamine beta-hydroxylase was suggested by incomplete correlation with plasma dopamine beta-hydroxylase (with an especially marked dissociation in renal disease) as well as the lack of a ventricular/lumbar cerebrospinal dopamine beta-hydroxylase concentration gradient. 3. Cerebrospinal fluid dopamine beta-hydroxylase was not changed by the central alpha 2-agonist clonidine at a dose that diminished cerebrospinal fluid noradrenaline, nor did cerebrospinal fluid dopamine beta-hydroxylase correspond between subjects to cerebrospinal fluid concentrations of noradrenaline or methoxyhydroxyphenylglycol; thus, cerebrospinal fluid dopamine beta-hydroxylase concentration was not closely linked either pharmacologically or biochemically to central noradrenergic neuronal activity. 4. Cerebrospinal fluid dopamine beta-hydroxylase was not changed in essential hypertension. In Parkinson's disease, cerebrospinal fluid dopamine beta-hydroxylase was markedly diminished (16.3 +/- 2.9 versus 31.3 +/- 1.4 ng/ml, P < 0.001) and rose by 58 +/- 21% (P = 0.02) after adrenal-to-caudate chromaffin cell autografts. In congenital dopamine beta-hydroxylase deficiency, lack of detectable dopamine beta-hydroxylase immunoreactivity in cerebrospinal fluid or plasma suggests absent enzyme (rather than a catalytically defective enzyme) as the origin of the disorder. 5. We conclude that cerebrospinal fluid dopamine beta-hydroxylase immunoreactivity, while not closely linked to central noradrenergic neuronal activity, is at least in part derived from the central nervous system, and that its measurement may be useful in both the diagnosis and treatment of neurological disease.