Increasing Progenitor Cell Proliferation in the Sub-Ventricular Zone: A Therapeutic Treatment for Progressive Multiple Sclerosis?

INTRODUCTION: The purpose of this study was to determine if pharmacological treatment could increase progenitor cell proliferation in the Sub-ventricular Zone of aged rats. Previous work had shown that increasing progenitor cell proliferation in this region correlated well (R2=0.78; p= 0.0007) with functional recovery in a damaged corpus callosum (white matter tract), suggesting that progenitor cell proliferation results in oligodendrocytes in this region. METHODS: 10 month old male and female Sprague Dawley rats were fed the drugs for 30 days in cookie dough, then immunocytochemistry was performed on coronal brain sections, using Ki67 labeling to determine progenitor cell proliferation. RESULTS: Female rats showed low endogenous (control) progenitor cell proliferation, significantly different from male rats (P<0.0001), at this age. Ascorbic Acid (20 mg/kg, daily for 30 days) increased progenitor cell proliferation overall, but maintained the innate gender difference in stem cell proliferation (P=0.001). Prozac (5 mg/kg, daily for 30 days) increased progenitor cell proliferation for females but decreased stem cell proliferation for males, again showing a gender difference (P<0.0001). Simvastatin (1 mg/kg for 30 days) also increased progenitor cell proliferation in females and decreased progenitor cell proliferation in males, leading to a significant gender difference. DISCUSSION: The three drug combinations (fluoxetine, simvastatin, and ascorbic acid, patent # 9,254,281) led to ~ 4 fold increase in progenitor cell proliferation in females, while male progenitor cell proliferation was highest with 50 mg/kg ascorbic acid. However, the ascorbic acid increase in proliferation appears to be only on the sides of the ventricles, which is not the region that normally gives rise to oligodendrocytes. CONCLUSION: There are innate gender differences in progenitor cell proliferation at the Sub-Ventricular Zone at middle age in rats, possibly due to the loss of estrogen in females. We also see notable gender differences in progenitor cell proliferation in the Sub ventricular Zone in response to common drugs, such as fluoxetine, simvastatin and Vitamin C (ascorbic acid).

Gender- and region-specific changes in estrogen signaling in aging rat brain mitochondria.

Recently epidemiological studies suggest females lose neuroprotection from neurodegenerative diseases as they go through menopause. It has been hypothesized that this neuroprotection is hormone-dependent. The current study characterized cell signaling molecules downstream of estrogen receptor beta that are known to play a role in memory, PKC, ERK, and connexin-43, in regions of the brain associated with memory decline in an attempt to elucidate significant changes that occur post-estrus. Total whole cell lysates were compared to isolated mitochondrial protein because mitochondrial function is known to be altered during aging. As hypothesized, protein concentrations differed depending on age, gender, and brain region. Additionally, many of these changes occurred within mitochondria but not within whole cell lysates indicating that these are epigenetic alterations. These findings accentuate the complexity of aging and provide insight into the gender-specific cellular processes that occur throughout this process.

Examining the Reversibility of Long-Term Behavioral Disruptions in Progeny of Maternal SSRI Exposure.

Serotonergic dysregulation is implicated in numerous psychiatric disorders. Serotonin plays widespread trophic roles during neurodevelopment; thus perturbations to this system during development may increase risk for neurodevelopmental disorders. Epidemiological studies have examined association between selective serotonin reuptake inhibitor (SSRI) treatment during pregnancy and increased autism spectrum disorder (ASD) risk in offspring. It is unclear from these studies whether ASD susceptibility is purely related to maternal psychiatric diagnosis, or if treatment poses additional risk. We sought to determine whether maternal SSRI treatment alone or in combination with genetically vulnerable background was sufficient to induce offspring behavior disruptions relevant to ASD. We exposed C57BL/6J or Celf6+/- mouse dams to fluoxetine (FLX) during different periods of gestation and lactation and characterized offspring on tasks assessing social communicative interaction and repetitive behavior patterns including sensory sensitivities. We demonstrate robust reductions in pup ultrasonic vocalizations (USVs) and alterations in social hierarchy behaviors, as well as perseverative behaviors and tactile hypersensitivity. Celf6 mutant mice demonstrate social communicative deficits and perseverative behaviors, without further interaction with FLX. FLX re-exposure in adulthood ameliorates the tactile hypersensitivity yet exacerbates the dominance phenotype. This suggests acute deficiencies in serotonin levels likely underlie the abnormal responses to sensory stimuli, while the social alterations are instead due to altered development of social circuits. These findings indicate maternal FLX treatment, independent of maternal stress, can induce behavioral disruptions in mammalian offspring, thus contributing to our understanding of the developmental role of the serotonin system and the possible risks to offspring of SSRI treatment during pregnancy.

Refining forelimb asymmetry analysis: Correlation with Montoya staircase contralateral function post-stroke.

BACKGROUND: Forelimb Asymmetry Test is a simple test of motor function, using exploration behavior of a rat in a novel environment and counting the number of times that a rat touches the wall with either forepaw. Our lab has noticed, however, that there appears to be an increased number of fingertip touches to the wall following a stroke in the impaired forelimb. NEW METHOD: We counted the number of times that the animal either laid its palm flat against the wall of the chamber or touched the wall with only its fingertips, for both the left and right forepaws. We also separated bouts of exploration, so we could clearly determine if fingertip touches normally were associated with a transition from resting state to exploration state. RESULTS AND COMPARISON WITH EXISTING METHODS: Fishers exact test indicated that there were significant differences in the way that the animals touched the wall pre-stroke compared to post-stroke, with more fingertip touches occurring post-stroke. Counting palm touches as normal and fingertip touches as abnormal increases the sensitivity of the Forelimb Asymmetry analysis and gives a good correlation with the contralateral functional deficits determined by Montoya Staircase post-stroke. If we counted every wall touch as normal (palm touches and fingertip touches), we see a loss of sensitivity and a poor correlation with contralateral function as determined by Montoya Staircase. CONCLUSIONS: This refinement of the Forelimb Asymmetry analysis improves correlation with Montoya Staircase contralateral function after stroke.

Increasing neurogenesis with fluoxetine, simvastatin and ascorbic Acid leads to functional recovery in ischemic stroke.

Less than 8.5% of ischemic stroke patients receive clot-busting drugs within the narrow time needed to reduce injury. Thus, there is need for an easily-accessible delayed post-stroke drug treatment to improve functional recovery. Various combinations of fluoxetine, simvastatin, and ascorbic acid were given to healthy rats to assess impact on neurogenesis versus controls. Fluoxetine combined with simvastatin and ascorbic acid produced a 19-fold increase in neurogenesis versus controls in healthy rats; fluoxetine alone produced 10-fold increase. We next tried a couple of drug combinations versus control in endothelin-induced stroked rats. Combined fluoxetine/ simvastatin/ascorbic acid treatment, given to stroked rats 20-26 hours after stroke induction and continued for 31 days, produced strong recovery as measured by Montoya staircase test (mean recovery to 85% of pre-stroke function) and Forelimb Asymmetry test (mean recovery to 90% of pre-stroke function). Fluoxetine and ascorbic acid without simvastatin only produced ~50% of recovery produced by the 3-drug combination. Our results indicate that combined treatment of Fluoxetine, simvastatin and ascorbic acid represents a promising delayed stroke treatment that greatly improves functional recovery in rats and warrants further study in human patient populations. This work formed the basis for a patent submission (US20130065924A1) Composition and method for treatment of neurodegeneration.

Genetic barcoding of marine leeches (Ozobranchus spp.) from Florida sea turtles and their divergence in host specificity.

Ozobranchus margoi and Ozobranchus branchiatus are the only two species of marine turtle leeches (Ozobranchus spp.) known to inhabit the Atlantic coast of the United States and the Gulf of Mexico. In early reports of fibropapillomatosis (FP) in green turtles (Chelonia mydas), O. branchiatus was implicated as a vector in the transmission of Fibropapilloma-associated turtle herpesvirus (FPTHV). It is imperative that the leech species be identified to elucidate the role Ozobranchus spp. may play in disease transmission. In this study, Ozobranchus branchiatus has been identified for the first time on a loggerhead (Caretta caretta) turtle, and the molecular data for this species is now available for the first time in GenBank. Both species of leeches were also found infecting a single C. mydas. Using morphological taxonomy combined with distance- and character-based genetic sequence analyses, this study has established a DNA barcode for both species of Ozobranchus spp. leech and has shown it can be applied successfully to the identification of leeches at earlier stages of development when morphological taxonomy cannot be employed. The results suggest a different haplotype may exist for O. branchiatus leeches found on C. caretta versus C. mydas. Leech cocoon residue collected from a C. mydas was identified using the new method.

Immunolocalization of NaV1.2 channel subtypes in rat and cat brain and spinal cord with high affinity antibodies.

High titer polyclonal antibodies were produced in rabbit against a peptide unique to NaV1.2 sodium channels. NaV1.2 antibodies displayed 500,000-fold greater affinity for the NaV1.2 peptide compared with NaV1.1 or NaV1.3 peptides from the same region. These antibodies, when coupled to Sepharose beads, retained saxitoxin binding sites from solubilized rat brain membranes. Eluted protein from this antibody-affinity column was recognized by antibodies directed against neuronal voltage-gated sodium channels. Rabbit antibodies, which had been partially purified, were used in immunocytochemical localization of the NaV1.2 channel in 50 microm rat brain slices at dilutions of 1:1000 or 1:2000. NaV1.2 channels were predominately localized in unmyelinated fibers in the cortex, hippocampus, spinal cord and hypothalamus. Varicosities were seen in fiber staining which may reflect true varicosities in the fiber or simply varying densities of sodium channels along the fiber. Cell body staining with the NaV1.2 antibody was primarily observed in the hypothalamus. Antibody staining in the cerebellum was complex, with staining observed primarily in posterior lobes and considerably lower amounts of staining observed in anterior lobes. Specific staining was limited to fibers located in the granule and molecular layer, in an orientation consistent with granule cell unmyelinated axon labeling.

Physiological characterization of the Na(+)/Ca(2+) exchanger (NCX) in hepatopancreatic and antennal gland basolateral membrane vesicles isolated from the freshwater crayfish Procambarus clarkii.

The purpose of this study was to physiologically characterize the basolateral Na(+)/Ca(2+) exchanger (NCX) in basolateral membrane vesicles (BLMVs) of hepatopancreas and antennal gland of intermolt crayfish. Conditions were optimized to measure Na(+)-dependent Ca(2+) uptake and retention in the BLMV including use of intravesicular (IV) oxalate and measuring initial uptake rates at 20 s. Na(+)-dependent Ca(2+) uptake rate into BLMV was temperature insensitive. Na(+)-dependent Ca(2+) uptake rate was dependent upon free Ca(2+) with saturable Michaelis-Menten kinetics determined as follows: hepatopancreas, maximal uptake rate (J(max))=2.45 nmol/mg per min, concentration at which carrier operates at half-maximal uptake rate (K(m))=0.69 microM Ca(2+); antennal gland, J(max)=13.2 nmol/mg per min, K(m)=0.59 microM Ca(2+). The two vesicle populations exhibited different sensitivity to putative NCX inhibitors. Benzamil had no effect on Na(+)-dependent Ca(2+) uptake rate in hepatopancreas; in antennal gland it was inhibitory at concentrations up to 30 microM and was stimulatory at higher concentrations. Conversely the inhibitor quinacrine was inhibitory at 10 microM in hepatopancreas and was stimulatory at 1000 microM; meanwhile it was ineffective in antennal gland BLMV. Short circuiting the BLMV had no effect on Na(+)-dependent Ca(2+) uptake rate suggesting that the process may be electroneutral. Compared with another prominent basolateral transporter in hepatopancreas the plasma membrane Ca(2+) ATPase (PMCA), the NCX has 70-fold greater J(max) (at comparable temperature) and a lower affinity. In antennal gland the NCX has 40-fold greater J(max) and a lower affinity. In hepatopancreas and antennal gland BLMV NCX appears to determine the rate of basolateral Ca(2+) efflux in intermolt.