The analgesic potential of intraventricular polymer-encapsulated adrenal chromaffin cells in a rodent model of chronic neuropathic pain.

Adrenal chromaffin cells reportedly produce analgesic effects when implanted in the periaqueductal gray and the intrathecal space near the spinal cord. Chromaffin cells implanted in the cerebral ventricles may also produce analgesic effects, and the availability of the cerebral ventricles as a potential implant site could be advantageous for some patients. In fact, some of the first patients were implanted in the intraventricular site, even though the analgesic potential of that site had never been demonstrated. The present study was conducted to assess the analgesic potential of intraventricular, polymer-encapsulated calf adrenal chromaffin cells in the Bennett model. Sciatic nerve ligations produced substantial, long-lasting pain-related behaviors. However, there was no evidence that polymer-encapsulated adrenal chromaffin cells implanted in the cerebral ventricles produce analgesic effects in this model of chronic neuropathic pain.

Generation and initial characterization of conditionally immortalized chromaffin cells.

Adrenal chromaffin cells have been successfully used to attenuate chronic pain when transplanted near the spinal cord, but primary cells are neither homogeneous nor practical for routine use in human therapy. Conditional immortalization with the temperature-sensitive allele of the large T antigen (tsTag) and creation of stable chromaffin cell lines would advance our understanding of both the use and limits of cell lines that contain this immortalization gene for such therapies. Cultures of embryonic day 17 rat adrenal and neonatal bovine adrenal cells were immortalized with the temperature-sensitive allele of SV40 tsTag and chromaffin cell lines established. The rat chromaffin line, RAD5.2, and the bovine chromaffin cell line, BADA.20, both expressed immunoreactivities (ir) for all the catecholamine enzymes: tyrosine hydroxylase (TH), the first enzyme in the synthetic pathway for catecholamines, dopa-beta-hydroxylase (DbetaH), and phenylethanolamine-N-methyltransferase (PNMT). At permissive temperature (33 degrees C), these chromaffin cells are proliferative, have a typical rounded chromaffinlike morphology, and contain detectable TH-, DbetaH-, and PNMT-ir. At nonpermissive temperature (39 degrees C), these cells stop proliferating, decrease Tag expression, and change the expression of TH-, DbetaH-, and PNMT-ir in vitro, suggesting increased differentiation at nonpermissive temperature. The chromaffin cell lines also express immunoreactivity for the opioid met-enkephalin (ENK) at permissive and nonpermissive temperatures. The expression of TH-ir in the bovine chromaffin cells is upregulated by the addition of dexamethasone (DEX) or forskolin during differentiation; TH-ir is not affected by the addition of DEX or forskolin in the rat chromaffin cells. The addition of forskolin during differentiation upregulates the expression of DbetaH-ir in the rat chromaffin cells. PNMT-ir is not affected by differentiation or agents in either cell line. However, catecholamine synthesis was not detectable by high-performance liquid chromatography, suggesting incomplete differentiation under current conditions, or influence by continued low levels of Tag expression. Both cell lines have been carried over many passages in vitro for more than 3 years and were repeatedly frozen and thawed. These data describe an initial step in the conditional immortalization of chromaffin cells that can maintain the phenotype of primary chromaffin cells in vitro over long periods. The use of such chromaffin cell lines that are able to deliver neuroactive molecules offers a novel approach to pain management.

Incomplete nigrostriatal dopaminergic cell loss and partial reductions in striatal dopamine produce akinesia, rigidity, tremor and cognitive deficits in middle-aged rats.

The present study was conducted to determine if the full array of parkinsonian symptoms could be detected in rats with nigrostriatal cell loss and striatal dopamine depletions similar to levels reported in the clinical setting, and to determine if older rats exhibit more robust parkinsonian deficits than younger rats. Young (2 months old) and middle-aged (12 months old) rats received bilateral striatal infusions of 6-OHDA, over the next 3 months they were assessed with a battery of behavioral tests, and then dopaminergic nigrostriatal cells and striatal dopamine and DOPAC levels were quantified. The results of the present study suggest that: (1) the full array of parkinsonian symptoms (i.e. akinesia, rigidity, tremor and visuospatial cognitive deficits) can be quantified in rats with incomplete nigrostriatal dopaminergic cell loss and partial reductions in striatal dopamine levels (2) parkinsonian symptoms were more evident in middle-aged rats with 6-OHDA infusions, and (3) there was evidence of substantial neuroplasticity in the older rats, but regardless of the age of the animal, endogenous compensatory mechanisms were unable to maintain striatal dopamine levels after rapid, lesion-induced nigrostriatal cell loss. These results suggest that using older rats with nigrostriatal dopaminergic cell loss and reductions in striatal dopamine levels similar to those in the clinical condition, and measuring behavioral deficits analogous to parkinsonian symptoms, might increase the predictive validity of pre-clinical rodent models.

Artificial neural network-aided image analysis system for cell counting.

BACKGROUND: In histological preparations containing debris and synthetic materials, it is difficult to automate cell counting using standard image analysis tools, i.e., systems that rely on boundary contours, histogram thresholding, etc. In an attempt to mimic manual cell recognition, an automated cell counter was constructed using a combination of artificial intelligence and standard image analysis methods. METHODS: Artificial neural network (ANN) methods were applied on digitized microscopy fields without pre-ANN feature extraction. A three-layer feed-forward network with extensive weight sharing in the first hidden layer was employed and trained on 1,830 examples using the error back-propagation algorithm on a Power Macintosh 7300/180 desktop computer. The optimal number of hidden neurons was determined and the trained system was validated by comparison with blinded human counts. System performance at 50x and lO0x magnification was evaluated. RESULTS: The correlation index at 100x magnification neared person-to-person variability, while 50x magnification was not useful. The system was approximately six times faster than an experienced human. CONCLUSIONS: ANN-based automated cell counting in noisy histological preparations is feasible. Consistent histology and computer power are crucial for system performance. The system provides several benefits, such as speed of analysis and consistency, and frees up personnel for other tasks.

Alleviation of behavioral deficits in aged rodents following implantation of encapsulated GDNF-producing fibroblasts.

The present study examined the effects of encapsulated cells which were genetically modified to secrete human glail-derived neurotrophic factor (hGDNF) on the motor deficits in aged rodents. Prior to implantation, animals were tested on a battery of motor tasks. Spontaneous locomotion and motor coordination was evaluated in young (5 month) and aged (20 months) rats. Aged animals tested for spontaneous locomotor activity were found to be hypoactive relative to young animals. Compared to the young animals the aged animals also: (1) were impaired on a bar pressing task, (2) were unable to descend a wooden pole covered with wire mesh in a coordinated manner, (3) fell more rapidly from a rotating rod and (4) were unable to maintain their balance on a series of wooden beams of varying widths. Following baseline testing, aged animals received either no implant, encapsulated baby hamster kidney fibroblast cells that were modified to produce hGDNF (BHK-hGDNF) or encapsulated BHK cells which were not modified to produce hGDNF (BHK-Control) implanted bilaterally into the striatum. Following surgery, a significant increase in locomotor activity and bar pressing was observed in those aged animals receiving BHK-hGDNF implants. Bar pressing in aged animals receiving BHK-Control cells was improved to a lesser extent and reached the level of performance seen in young rats. No recovery was observed in the animals receiving BHK-Control cell-loaded capsules on any of the other motor tasks. Histological analysis revealed that implants of hGDNF-producing cells produced a marked increase in the density of tyrosine hydroxylase staining in the striatum adjacent to the implant site. This increased staining was not seen in animals receiving BHK-Control cells. Histological analysis also revealed the presence of viable BHK-hGDNF cells within the capsules that continued to produce hGDNF as measured by ELISA. These results indicate that polymer-encapsulated hGDNF-secreting cells survive following implantation into aged rats and may be useful for treating some of the behavioral consequences of aging or disorders characterized by dopaminergic hypofunction.

Implants of encapsulated human CNTF-producing fibroblasts prevent behavioral deficits and striatal degeneration in a rodent model of Huntington's disease.

Delivery of neurotrophic molecules to the CNS has gained considerable attention as a potential treatment strategy for neurological disorders. In the present study, a DHFR-based expression vector containing the human ciliary neurotrophic factor (hCNTF) was transfected into a baby hamster kidney fibroblast cell line (BHK). Using a polymeric device, encapsulated BHK-control cells and those secreting hCNTF (BHK-hCNTF) were transplanted unilaterally into the rat lateral ventricle. Twelve days later, the same animals received unilateral injections of quinolinic acid (QA; 225 nmol) into the ipsilateral striatum. After surgery, animals were behaviorally tested for apomorphine-induced rotation behavior and for skilled forelimb function using the staircase test. Rats receiving BHK-hCNTF cells rotated significantly less than animals receiving BHK-control cells. No behavioral effects of hCNTF were observed on the staircase test. Nissl-stained sections demonstrated that BHK-hCNTF cells significantly reduced the extent of striatal damage produced by QA. Quantitative analysis of striatal neurons further demonstrated that both choline acetyltransferase- and GAD-immunoreactive neurons were protected by BHK-hCNTF implants. In contrast, a similar loss of NADPH-diaphorase-positive cells was observed in the striatum of both implant groups. Analysis of retrieved capsules revealed numerous viable and mitotically active BHK cells that continued to secrete hCNTF. These results support the concepts that implants of polymer-encapsulated hCNTF-releasing cells can be used to protect striatal neurons from excitotoxic damage and that this strategy may ultimately prove relevant for the treatment of Huntington's disease.