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.

Dissociable long-term cognitive deficits after frontal versus sensorimotor cortical contusions.

Cognitive deficits are the most enduring and disabling sequelae of human traumatic brain injury (TBI), but quantifying the magnitude, duration, and pattern of cognitive deficits produced by different types of TBI has received little emphasis in preclinical animal models. The objective of the present study was to use a battery of behavioral tests to determine if different impact sites produce different patterns of behavioral deficits and to determine how long behavioral deficits can be detected after TBI. Prior to surgery, rats were trained to criteria on delayed nonmatching to position, radial arm maze, and rotarod tasks. Rats received sham surgery (controls), midline frontal contusions (frontal TBI, 2.25 m/sec impact), or unilateral sensorimotor cortex contusions (lateral TBI, 3.22 m/sec impact) at 12 months of age and were tested throughout the next 12 months. Cognitive deficits were more robust and more enduring than sensorimotor deficits for both lateral TBI and frontal TBI groups. Lateral TBI rats exhibited transient deficits in the forelimb placing and in the rotarod test of motor/ambulatory function, but cognitive deficits were apparent throughout the 12-month postsurgery period on tests of spatial learning and memory including: (1)reacquisition of a working memory version of the radial arm maze 6-7 months post-TBI, (2) performance in water maze probe trials 8 months post-TBI, and (3) repeated acquisition of the Morris water maze 8 and 11 months post-TBI. Frontal TBI rats exhibited a different pattern of deficits, with the most robust deficits in tests of attention/orientation such as: (1) the delayed nonmatching to position task (even with no delays) 1-11 weeks post-TBI, (2) the repeated acquisition version of the water maze--especially on the first "information" trial 8 months post-TBI, (3) a test of sensorimotor neglect or inattention 8.5 months post-TBI, and (4) a DRL20 test of timing and/or sustained attention 11 months after surgery. These results suggest that long-term behavioral deficits can be detected in rodent models of TBI, that cognitive deficits seem to be more robust than sensorimotor deficits, and that different TBI impact sites produce dissociable patterns of cognitive deficits in rats.

Intraventricular encapsulated calf adrenal chromaffin cells: viable for at least 500 days in vivo without detectable adverse effects on behavioral/cognitive function or host immune sensitization in rats.

Numerous studies have reported that adrenal chromaffin cell transplants, including encapsulated xenogeneic adrenal chromaffin cells, have analgesic effects. However, in addition to efficacy, the clinical utility of encapsulated xenogeneic adrenal chromaffin cells for treatment of chronic pain is dependent on the duration of cell viability in vivo, and their relative safety. The objectives of the present study in rats were to: (1) examine encapsulated calf adrenal chromaffin (CAC) cells for evidence of viable cells and continued release of analgesic agents after an extended period in vivo; (2) determine if intraventricular encapsulated CAC cells produce detectable adverse effects on behavioral/cognitive function; and (3) test for evidence of host immune sensitization after an extended period of exposure to encapsulated xenogeneic adrenal chromaffin cells. Results of the present study suggest that some encapsulated CAC cells remain viable for nearly 1.5 years in vivo and continue to produce catecholamines and met-enkephalin. Post-explant device norepinephrine output was equivalent to amounts previously shown to produce analgesic effects with intrathecal implants. Encapsulated adrenal chromaffin cells also appeared relatively safe, even when implanted in the cerebral ventricals, with a lower side-effect profile than systemic morphine (4 mg/kg). There was no evidence that encapsulated CAC-cells implanted in the ventricles affected body weight, spontaneous activity levels, or performance in the delayed matching to position operant task which is sensitive to deficits in learning, memory, attention, motivation, and motor function. Finally, encapsulated CAC cells produced no detectable evidence of host immune sensitization after 16.7 months in vivo, although unencapsulated CAC cells produced a robust immune response even in aged rats. The results of the present study suggest that adrenal chromaffin cells remain viable in vivo for long periods of time, and that long-term exposure to encapsulated xenogeneic adrenal chromaffin cell implants appears relatively safe.

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.

Effects of intraventricular encapsulated hNGF-secreting fibroblasts in aged rats.

Exogenous NGF administered into the central nervous system (CNS) has been reported to improve cognitive function in aged rats. However, concerns have been expressed about the risks involved with supplying NGF to the CNS. In this study, baby hamster kidney cells (BHK) genetically modified to secrete human NGF (hNGF) were encapsulated in semipermeable membranes and implanted intraventricularly. ChAT/LNGFR-positive basal forebrain neurons were shown to atrophy and degenerate with age, especially in cognitively impaired rats. The encapsulated BHK-NGF cells produced less than 10% of doses previously reported to be effective, but this was sufficient to increase the size of ChAT/LNGFR-positive basal forebrain neurons in the aged and learning-impaired rats to the size of the neurons in young healthy rats. The hNGF from these encapsulated cells also improved performance in a repeated-acquisition version of the Morris water maze spatial learning task in learning-impaired 20.6- and 26.7-mo-old rats. Furthermore, there was no evidence that these doses of hNGF impaired Morris water maze performance in the youngest 3.3-5.4 mo rats, and analyses of mortality rates, body weights, somatosensory thresholds, potential hyperalgesia, and activity levels, suggested that these levels of exogenous hNGF are not toxic or harmful to aged rats. These results suggest that CNS-implanted semipermeable membranes, containing genetically modified xenogeneic cells continuously producing these levels of hNGF, attenuate age-related cognitive deficits in nonimmunosuppressed aged rats, and that both the surgical implantation procedure and long-term exposure to low doses of hNGF appear safe in aged rats.

Implantation of encapsulated catecholamine and GDNF-producing cells in rats with unilateral dopamine depletions and parkinsonian symptoms.

Studies in rodents suggest that PC12 cells, encapsulated in semipermeable ultrafiltration membranes and implanted in the striatum, have some potential efficacy for the treatment of age- and 6-OHD-induced sensorimotor impairments (22, 70, 71, 74). The objectives of this study were to: (1) determine if baby hamster kidney cells engineered to secrete glial cell line-derived neurotrophic factor (BHK-GDNF) would survive encapsulation and implantation in a dopamine-depleted rodent striatum, (2) compare polymer-encapsulated PC12 and PC12A cells in terms of their ability to survive and produce catecholamines in vivo in a dopamine-depleted striatum, and (3) determine if BHK-GDNF, PC12, or PC12A cells reduce parkinsonian symptoms in a rodent model of Parkinson's disease. Capsules with BHK-GDNF or PC12 cells contained viable cells after 90 days in vivo, with little evidence of host tissue damage/gliosis. In rats with tyrosine hydroxylase (TH)-positive fibers remaining in the lesioned striatum, there was TH-positive fiber ingrowth into the membranes of the BHK-GDNF capsules. PC12-containing capsules had higher basal release of both dopamine and L-DOPA after 90 days in vivo than before implantation, while basal release of both dopamine and L-DOPA decreased in the PC12A-containing capsules. Both encapsulated PC12 and PC12A cells, but not encapsulated BHK-GDNF cells, decreased apomorphine-induced rotations. Parkinsonian symptoms (akinesia, freezing/bracing, sensorimotor neglect) related to the extent of dopamine depletion were evident even in rats with dopamine depletions of only 25%. Evidence that encapsulated cells may attenuate these parkinsonian symptoms was not detected but most of the rats were more severely depleted of dopamine than Parkinson's patients (less than 2% dopamine remaining in the entire striatum), and these tests were not sensitive to differences between rats with less than 10% dopamine remaining. These results suggest that cell encapsulation technology can safely provide site-specific delivery of dopaminergic agonists or growth factors within the CNS, without requiring suppression of the immune system, and without using fetal tissue. Of the three types of encapsulated cells examined in the present study, PC12 cells seem to offer the most therapeutic potential in rats with severe dopamine depletions.

Recent progress in immunoisolated cell therapy.

Biohybrid implants represent a new class of medical device in which living cells, supported in a hydrogel matrix, and surrounded by a semipermiable membrane, produce and deliver therapeutic reagents to specific sites within a host. First proposed in the mid-1970s for diabetes, this treatment modality has progressed rapidly in the past four years and is now being investigated not just for endocrine disorders but also for alleviation of chronic pain, treatment of neurodegenerative disorders, and delivery of neurotrophic factors to sites within the blood brain barrier, and as a practical alternative to conventional ex vivo.

Polymer-encapsulated PC12 cells promote recovery of motor function in aged rats.

The feasibility of using polymer-encapsulated PC12 cells to ameliorate the motor deficits in aged rats was evaluated. Spontaneous locomotion and motor coordination was evaluated in young (5-6 month) and aged (24-25 month) rats. Aged animals tested for spontaneous locomotor activity in Digiscan animal activity monitors were found to be hypoactive relative to young animals. Compared to the young animals the aged animals: (1) remained suspended from a horizontal wire for less time, (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 with either a square or rounded top of varying widths. Prior to implantation PC12 cell-loaded capsules were chromatographically characterized for catecholamine release. Following baseline testing, aged animals received either no implant, empty capsules, or PC12 cell-loaded capsules implanted bilaterally into the striatum. Three weeks following surgery, animals were retested and a significant improvement in balance on the rotorod and wooden beams was observed in those aged animals receiving PC12 cell-loaded capsules. No recovery was observed in the animals receiving PC12 cell-loaded capsules on any of the other motor tasks. Likewise, no improvement was observed on any behavioral measure in those animals receiving empty capsules. Histological analysis revealed the presence of numerous surviving tyrosine hydroxylase-positive PC12 cells within the capsules. Encapsulated PC12 cells survive following implantation into aged rats and such a technique may be useful for treating some of the behavioral consequences of aging.

Locomotion of aged rats: relationship to neurochemical but not morphological changes in nigrostriatal dopaminergic neurons.

Spontaneous locomotion and motor coordination was evaluated in young (5-6 month old) and aged (24-25 month old) rats. Animals were tested for spontaneous locomotor activity in Digiscan Animal Activity Monitors during the nocturnal cycle. Aged animals exhibited a significant hypoactivity compared to their young counterparts. Evaluation of the time course of activity revealed that the young animals had a cyclical pattern of activity during the 12-hour testing period with clear peaks at 2-4 hours after the initiation of testing and at 8- to 10-hour intervals thereafter. In contrast, the aged animals exhibited a blunted initial activity peak. During the remainder of the test period the aged animals activity was stable with no further peaks in activity. Compared to the young animals the aged animals also (a) remained suspended from a horizontal wire for less time, (b) were unable to descend a wooden pole covered with wire mesh in a coordinated manner, (c) fell more rapidly from a rotating rod and (d) were unable to maintain their balance on a series of wooden beams with either a square or rounded top of varying widths. Histological analysis demonstrated that there was no reduction in the number, area, or length of tyrosine hydroxylase-immunoreactive neurons within the A8, A9, or A10 region of the aged animals. Neurochemical analysis revealed that while DA and HVA levels were not decreased in the aged rats, DOPAC levels, as well as the ratios of DA/DOPAC and DA/HVA, were decreased. These results indicate that neurochemical but not morphological changes within the nigrostriatal dopaminergic system underlie the deficits in motor behavior observed in aged rats.