Daniel A. Castellanos

TrkC overexpression enhances survival and migration of neural stem cell transplants in the rat spinal cord.

Although CNS axons have the capacity to regenerate after spinal cord injury when provided with a permissive substrate, the lack of appropriate synaptic target sites for regenerating fibers may limit restoration of spinal circuitry. Studies in our laboratory are focused on utilizing neural stem cells to provide new synaptic target sites for regenerating spinal axons following injury. As an initial step, rat neural precursor cells genetically engineered to overexpress the tyrosine kinase C (trkC) neurotrophin receptor were transplanted into the intact rat spinal cord to evaluate their survival and differentiation. Cells were either pretreated in vitro prior to transplantation with trkC ligand neurotrophin-3 (NT-3) to initiate differentiation or exposed to NT-3 in vivo following transplantation via gelfoam or Oxycel©. Both treatments enhanced survival of trkC-overexpressing stem cells to nearly 100%, in comparison with approximately 30–50% when either NT-3 or trkC was omitted. In addition, increased migration of trkC-overexpressing cells throughout the spinal gray matter was noted, particularly following in vivo NT-3 exposure. The combined trkC expression and NT-3 treatment appeared to reduce astrocytic differentiation of transplanted neural precursors. Decreased cavitation and increased β-tubulin fibers were noted in the vicinity of transplanted cells, although the majority of transplanted cells appeared to remain in an undifferentiated state. These findings suggest that genetically engineered neural stem cells in combination with neurotrophin treatment may be a useful addition to strategies for repair of spinal neurocircuitry following injury.

Amiodarone-induced delirium.

Excerpt Psychiatric disturbances have not been associated with amiodarone therapy (1, 2). We report the case of a patient who had an acute episode of delirium while taking amiodarone. The delirium ...

Improved neural progenitor cell survival when cografted with chromaffin cells in the rat striatum.

Transplantation of stem and neural progenitor cells hold great promise in the repair of neuronal tissue lost due to injury or disease. However, survival following transplantation to the adult CNS has been poor, likely due to a lack of neurotrophic factors, such as basic fibroblast growth factor (FGF-2), that are used to maintain and expand these cells in culture. Chromaffin cells produce several neurotrophic agents, including FGF-2, which may aid in both neuroprotection following injury and progenitor cell proliferation and survival. In addition, increased CNS catecholamines have been shown to improve functional recovery following insult. Thus, cotransplants of neural progenitor cells and chromaffin cells may be a useful clinical strategy. To address this, the survival of rat cortical progenitors transplanted to the adult rat striatum with and without bovine chromaffin cell cografts was assessed. Progenitors obtained from E14 embryos were prelabeled with bromodeoxyuridine (BrdU) before transplantation to enable later identification. Transplants were made both unilaterally and bilaterally, where animals received a monograft (progenitor cells alone) on one side and a cograft (progenitors + chromaffin cells) on the other. Histological results after 7, 17, and 30 days posttransplant revealed greatly improved survival of BrdU-labeled cells in the cografts and also less infiltration of presumptive immune cells. In addition, perivascular cuffing was seen in the monografts. In vitro progenitor cohorts stained positive for nestin, GFAP, and beta-tubulin III, but in vivo very few cells were found that were double labeled with BrdU and one of these markers. Thus, in contrast to in vitro findings, chromaffin cells did not enhance differentiation of progenitors in vivo during the 30 days posttransplantation. The results of these studies suggest that chromaffin cells may provide neurotrophic support to enhance survival, but not differentiation, of cortical progenitor grafts in the adult CNS.

Sustained analgesic peptide secretion and cell labeling using a novel genetic modification

Cell-based therapy for neuropathic pain could provide analgesics to local pain modulatory regions in a sustained, renewable fashion. In order to provide enhanced analgesic efficacy, transplantable cells may be engineered to produce complementary or increased levels of analgesic peptides. In addition, genetic labeling of modified cells is desirable for identification and tracking, but it should be retained intracellularly as desired analgesic peptides are secreted. Usually constructs encode proteins destined for either extra- or intracellular compartments, as these pathways do not cross. However, interactions between intracellular destinations provide a window of opportunity to overcome this limitation. In this report, we have explored this approach using a potential supplementary analgesic peptide, [Ser1]-histogranin (SHG), the stable synthetic derivative of a naturally occurring peptide with N-methyl D-aspartate (NMDA) antagonistic properties. A synthetic SHG gene was combined with (i) nerve growth factor-β (NGF-β) amino-terminal signal peptide to enable secretion, and (ii) a fluorescent cellular label (mRFP) with intervening cathepsin L cleavage site, and subcloned into a lentiviral vector. In addition, an endoplasmic retention signal, KDEL, was added to enable retrieval of mRFP. Using immunocytochemistry and confocal microscopic profile analysis, cells transduced by such lentiviruses were shown to synthesize a single SHG-mRFP polypeptide that was processed, targeted to expected subcellular destinations in several cell types. Dot blot and Western analysis revealed stable transduction and long-term secretion of SHG from PC12 cells in vitro. Transplantation of such cells provided modest analgesia in a rodent pain model consistent with low levels of SHG peptide in the cerebrospinal fluid (CSF). These results suggest that it is possible to deliver proteins with different final destinations from a single construct, such as pharmacologically active peptide for secretion and intracellular label for identifying transplantable cells.

Prolonged nociceptive responses to hind paw formalin injection in rats with a spinal cord injury

Unilateral lesioning of the spinal dorsal horn with the excitotoxin quisqualic acid (QUIS) leads to robust degeneration of dorsal horn grey matter, and robust pain-related symptoms, such as cutaneous hypersensitivity, persist long after injury. A possible mechanism that underlies the pain-related symptoms is the disruption of dorsal horn inhibitory neuron function, leading to decreased inhibition of nociceptive neurons. Five percent formalin was injected into the hind paw of rats with either a QUIS lesion or sham lesion. Both QUIS- and sham-lesioned rats displayed bi-phasic hind paw flinches following formalin injection, but a prolonged response was observed in QUIS-lesioned rats. The expression of the immediate-early gene product Fos in the dorsal horn ipsilateral to formalin injection was similar between QUIS- and sham-lesioned rats. In QUIS-lesioned rats, however, there was a marked absence of dorsal horn neurons, particularly GABAergic neurons, compared to sham-lesioned rats. The prolonged nociceptive response observed with a unilateral QUIS lesion may be due to generalized changes in dorsal horn neuron function including a loss of inhibitory neuron function.

Expansion of formalin-evoked Fos-immunoreactivity in rats with a spinal cord injury

Peripheral tissue injury as well as spinal cord injury (SCI) may lead to sensitization of dorsal horn neurons and alterations in nociceptive processing. Thus, peripheral injuries experienced by SCI patients, even if not initially perceived, could result in a persistent and widespread activation of dorsal horn neurons and emerge as chronic pain with interventive repair or modest recovery from SCI. To visualize the spinal neuron response to peripheral tissue injury following complete SCI in rats, the neural transcription factor Fos was quantitated in the spinal cord. Two weeks following either a complete transection of the spinal cord at the level of T8 or a sham surgery (laminectomy), rats were injected with formalin into the left hind paw. Sham-operated rats demonstrated biphasic hind paw pain-related behavior following formalin injection, but transected rats displayed fewer behaviors in the second (tonic) phase. Stereological analysis of the sham group revealed that the extent of formalin-induced Fos expression was within the lumbar dorsal horn, with numerous Fos-like immunoreactive profiles in the ipsilateral dorsal horn and some contralateral immunoreactive profiles. In contrast, the level of Fos-like immunoreactivity in the transected group was significantly elevated and expanded in range compared to the sham group, with increases observed in the normal laminar distribution regions, as well as multi-segmentally through sacral levels and increases in the contralateral dorsal horn segments. The data demonstrate that widespread activation of spinal, especially dorsal horn, neurons following peripheral insult can occur in the injured spinal cord, despite reduced pain responsiveness, and suggests that exaggerated pain may emerge as spinal recovery or repair progresses.

YOUTH SUICIDE AND HURRICANE ANDREW

Journal of the American Academy of Child & Adolescent Psychiatry - Vol. 42 - N° 2 - p. 131

Antinociceptive effects of topical mepivacaine in a rat model of HIV-associated peripheral neuropathic pain.

Background A consequence of HIV infection is sensory neuropathy, a debilitating condition that degrades the quality of life of HIV patients. Furthermore, life-extending antiretroviral treatment may exacerbate HIV sensory neuropathy. Analgesics that relieve other neuropathic pains show little or no efficacy in ameliorating HIV sensory neuropathy. Thus, there is a need for analgesics for people with this particular pain. While lidocaine is used in the management of painful peripheral neuropathies, another local anesthetic mepivacaine, with a potentially improved bioavailability, could be utilized for the management of HIV neuropathic pain. Methods The efficacy of topical anesthetics was evaluated in a preclinical rodent model of painful peripheral neuropathy induced by epineural administration of the HIV envelope protein gp120 delivered using saturated oxidized cellulose implanted around the sciatic nerve. Beginning at 2 weeks following gp120 administration, the effects of local anesthetics topically applied via gauze pads were tested on heat and mechanical hyperalgesia in the hind paw. Rats were tested using several concentrations of mepivacaine or lidocaine during the following 2 weeks. Results By 2 weeks following epineural gp120 implantation, the ipsilateral hind paw developed significant hypersensitivity to noxious pressure and heat hyperalgesia. A short-lasting, concentration-dependent amelioration of pressure and heat hyperalgesia was observed following topical application of mepivacaine to the ipsilateral plantar hind paw. By contrast, topical lidocaine ameliorated heat hyperalgesia in a concentration-dependent manner but not pressure hyperalgesia. Equipotent concentrations of mepivacaine and lidocaine applied topically to the tail of mice significantly increased tail withdrawal latencies in the tail flick test, demonstrating that both local anesthetics attenuate responding to a brief noxious stimulus. Conclusion These findings showed that mepivacaine, rather than lidocaine, consistently attenuated two distinct symptoms of neuropathic pain and suggest that topical formulations of this local anesthetic could have utility in the alleviation of clinical HIV neuropathic pain.

Coupling estimation and compensation model for arrays of multimode radiators

In the design process of array antennas, coupling is one of the most important aspects to be considered to complete a realistic model. The real feeding radiated coefficients can be quite different from the theoretical ones because of this effect. A characterization and compensation model is presented. This procedure is able to match each element from the array. All the parameters of the array coupling model are obtained through scattering parameters and radiation pattern measurements. Some practical applications to linear patch arrays are presented.