Uninjected Side Research Articles

The effect of preoperative botulinum toxin a injection on traction force during hernia repair: a prospective, single-blind study, intra-patient comparison using contralateral side as a control

PurposeVentral hernias are a common complication of laparotomy, posing challenges particularly when primary fascial closure is unattainable. Although chemical component separation using preoperative botulinum toxin A (BTX) injections has emerged as a promising adjunct, objective evidence of its efficacy remains limited. This study aimed to objectively assess the effect of preoperative BTX on traction force during ventral hernia repair.MethodsA prospective, single-blind study was conducted on patients with midline incisional hernias following liver transplantation. BTX was administered unilaterally, and the traction force required to medially advance the anterior rectus sheath was measured intraoperatively. Pre- and post-injection CT scans were analyzed for changes in hernia size and LAW muscle measurements. Statistical analyses were performed to evaluate traction force differences between BTX-injected and uninjected sides.ResultsTen patients underwent hernia repair with primary fascial closure achieved in all cases. Comparison of pre- and post-injection CT scans showed no significant changes in hernia size. LAW muscle length increased by 1.8 cm, while thickness decreased by 0.2 cm. Intraoperative traction force measurements revealed a significant reduction on the BTX-injected side compared to the uninjected side (p < 0.0001). The traction force ratio on the BTX-injected to the uninjected side averaged 57%, indicating the efficacy of BTX in reducing tension.ConclusionPreoperative BTX significantly reduces traction force during ventral hernia repair, highlighting its potential as an adjunctive therapy in complex cases. While challenges remain in patient selection and outcome assessment, BTX offers a promising avenue for enhancing abdominal wall reconstruction outcomes and reducing surgical complications.

Influences of GABAergic Inhibition in the Dorsal Medulla on Contralateral Swallowing Neurons in Rats

We aimed to examine the effect of unilateral inhibition of the medullary dorsal swallowing networks on the activities of swallowing-related cranial motor nerves and swallowing interneurons. In 25 juvenile rats, we recorded bilateral vagal nerve activity (VNA) as well as unilateral phrenic and hypoglossal activity (HNA) during fictive swallowing elicited by electrical stimulation of the superior laryngeal nerve during control and following microinjection of the GABA agonist muscimol into the caudal dorsal medulla oblongata in a perfused brainstem preparation. In 20 animals, swallowing interneurons contralateral to the muscimol injection side were simultaneously recorded extracellularly and their firing rates were analyzed during swallowing. Integrated VNA and HNA to the injection side decreased to 49.0 ± 16.6% and 32.3 ± 17.9%, respectively. However, the VNA on the uninjected side showed little change after muscimol injection. Following local inhibition, 11 out of 20 contralateral swallowing interneurons showed either increased or decreased of their respective firing discharge during evoked-swallowing, while no significant changes in activity were observed in the remaining nine neurons. The neuronal networks underlying the swallowing pattern generation in the dorsal medulla mediate the ipsilateral motor outputs and modulate the contralateral activity of swallowing interneurons, suggesting that the bilateral coordination of the swallowing central pattern generator regulates the spatiotemporal organization of pharyngeal swallowing movements. NA Laryngoscope, 131:2187-2198, 2021.

Comparison of preseptal and pretarsal onabotulinum toxin an injection in patients with hemifacial spasm

Aim The aim of the present study was to evaluate the effects of different doses of onabotulinum toxin A on the amplitude and latency values of the blink reflex and facial nerve in the pretarsal and preseptal portions of the orbicularis oculi muscle in patients with hemifacial spasm. Materials and methods Thirty patients with hemifacial spasm were assigned in two equal groups: Pretarsal Group: Five units of onabotulinum toxin A were injected into each of 2 points of the pretarsal portion; Preseptal Group: Five units of onabotulinum toxin A was injected into 4 points of the preseptal portion. We compared the electromyographic features of the patients before and 5 weeks after botulinum toxin (BTX) injection. Results In comparison of pre- and post-treatment measurements of blink reflex amplitude responses, the decreases in R1 (p = 0.003), R2 (p < 0.001), and R2C amplitudes (p = 0.031) were found to be significant in the BTX injected side in the pretarsal group. In the comparison of pre- and post-treatment measurements of facial nerve compound action potential amplitude changes, decreases in the amplitudes of the BTX injected (ipsilateral), and uninjected (contralateral) side in the pretarsal group were found to be significant (p < 0.001 for both groups). Decreases in the amplitudes of the BTX injected, and uninjected side in the preseptal group were found to be significant (p < 0.001, and p = 0.008, respectively). Conclusion According to our hypothesis, the smaller amount of BTX applied to the pretarsal portion was found to be more effective than higher amount of BTX injected into the preseptal portion of the orbicularis oculi muscle.

Pathological ASXL1 Mutations and Protein Variants Impair Neural Crest Development.

SummaryThe neural crest (NC) gives rise to a multitude of fetal tissues, and its misregulation is implicated in congenital malformations. Here, we investigated molecular mechanisms pertaining to NC-related symptoms in Bohring-Opitz syndrome (BOS), a developmental disorder linked to mutations in the Polycomb group factor Additional sex combs-like 1 (ASXL1). Genetically edited human pluripotent stem cell lines that were differentiated to NC progenitors and then xenotransplanted into chicken embryos demonstrated an impairment of NC delamination and emigration. Molecular analysis showed that ASXL1 mutations correlated with reduced activation of the transcription factor ZIC1 and the NC gene regulatory network. These findings were supported by differentiation experiments using BOS patient-derived induced pluripotent stem cell lines. Expression of truncated ASXL1 isoforms (amino acids 1–900) recapitulated the NC phenotypes in vitro and in ovo, raising the possibility that truncated ASXL1 variants contribute to BOS pathology. Collectively, we expand the understanding of truncated ASXL1 in BOS and in the human NC.

Feto-Maternal Trafficking of Exosomes in Murine Pregnancy Models.

Timing and initiation of labor are well-orchestrated by signals communicated between the fetal and maternal compartments; however, how these signals are communicated is not completely understood. Fetal exosomes, intercellular signaling vesicles, may play a key role in the process. The objective of this study was to evaluate exosome trafficking in vivo from fetal to maternal compartments. Pregnant CD-1 mice were intra-amniotically injected on gestational day 16 and 17 with exosomes isolated from primary human amnion epithelial cells fluorescently labeled with the lipophilic dye 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR). All our analyses were performed on samples collected on Day 18. After 24 h, mice were imaged using Bruker MS FX PRO In vivo Imager and tissues were collected. In vivo imaging of mouse showed fluorescence in the uterus, on the exosome-injected side whereas the uterine tissues from the uninjected side and saline and dye alone injected animals remained negative. Histological analysis of placenta showed exosome migration from the fetal to the maternal side of the placenta. Fluorescence released from exosomes was seen in maternal blood samples as well as in maternal uterus and kidneys. This study demonstrates that exosomal cargo can be carried through systemic route from the fetal to the maternal side of the uterine tissues during pregnancy, supporting the idea that fetal signals can be delivered via exosomes.

Bone and cartilage changes in rabbit mandibular condyles after 1 injection of botulinum toxin.

Temporary paralysis of the masseter muscle caused by botulinum toxin is a common treatment for temporomandibular disorders, bruxism, and muscle hypertrophy. Loss of masseter force is associated with decreased mandibular mineral density. Our objectives were (1) to establish whether bone loss at the mandibular condyle is regionally specific and (2) to ascertain whether the treatment affects the condylar cartilage. Young adult female rabbits received a unilateral masseter injection of botulinum neurotoxin serotype A (BoNT/A, n = 31), saline solution (n = 19), or no injection (n = 3) and were also injected with bromodeoxyuridine (BrdU), a replication marker. The rabbits were killed at 4 or 12 weeks after treatment. The condyles were processed for paraffin histology. Cortical thickness, cartilage thickness, and trabecular bone areal density were measured, and replicating cells were counted after BrdU reaction. The BoNT/A rabbits exhibited a high frequency of defects in the condylar bone surface, occurring equally on the injected and uninjected sides. Bone loss was seen only on the side of the BoNT/A injection. Cortical as well as trabecular bone was severely affected. The midcondylar region lost the most bone. Recovery at 12 weeks was insignificant. Condylar cartilage thickness showed no treatment effect but did increase with time. The numbers of proliferating cells were similar in the treatment groups, but the BoNT/A animals showed more side asymmetry associated with the condylar defects. Bone loss may be a risk factor for the use of botulinum toxin in jaw muscles.

Ultrasound-Guided Greater Occipital Nerve Block: An Efficient Technique in Chronic Refractory Migraine Without Aura?

Background: The effectiveness of greater occipital nerve block (GONB) in patients with primary headache syndromes is controversial. Few studies have been evaluated the usefulness of GONB in patients with migraine without aura (MWOA). Objective: To compare the effectiveness of ultrasound-guided GONB using bupivacaine 0.5% and placebo on clinical improvement in patients with refractory MWOA in a randomized, doubleblinded clinical trial. Study Design: A prospective, randomized, placebo-controlled, double-blind pilot trial. Setting: Physical medicine and rehabilitation and neurology departments of a University Hospital. Methods: Thirty-two patients with a diagnosis of MWOA according to the International Classification of Headache Disorders-II criteria were included in the study. Twenty-three patients (2 men, 21 women) completed the study. They were randomly assigned to receive either GONB with local anesthetic (bupivacaine 0.5% 1.5 mL) or greater occipital nerve (GON) injection with normal saline (0.9% 1.5 mL). Ultrasound-guided GONB was performed to more accurately locate the nerve. All procedures were performed using a 7 – 13 MHz high-resolution linear ultrasound transducer. The treatment group was comprised of 11 patients and the placebo group was comprised of 12 patients. The primary outcome measure was the change in the headache severity score during the one-month post-intervention period. Headache severity was assessed with a visual analogue scale (VAS) from 0 (no pain) to 10 (intense pain). Results: In both groups, a decrease in headache intensity on the injection side was observed during the first post-injection week and continued until the second week. After the second week, the improvement continued in the treatment group, and the VAS score reached 0.97 at the end of the fourth week. In the placebo group after the second week, the VAS values increased again and nearly reached the pre-injection levels. The decrease in the monthly average pain intensity score on the injected side was statistically significant in the treatment group (P = 0.003), but not in the placebo group (P = 0.110). No statistically significant difference in the monthly average pain intensity score was observed on the uninjected side in either group (treatment group, P = 0.994; placebo group, P = 0.987). No serious side effect was observed after the treatment in either group. Only one patient had a self-limited vaso-vagal syncope during the procedure. Limitations: This trial included a relatively small sample. This may have been the result of the inclusion of only those patients who correctly completed their pain diaries. Another major limitation is the short follow-up duration. Patients were followed for one month after the injection, thus relatively long-term effects of the injection have not been observed. Conclusions: Ultrasound guided GONB with 1.5 mL of 0.5% bupivacaine for the treatment of migraine patients is a safe, simple, and effective technique without severe adverse effects. To increase the effectiveness of the injection, and to implement the isolated GONB, ultrasonography guidance could be suggested. Key words: Migraine, greater occipital nerve, occipital nerve block, ultrasound-guided

Inclusion of a portion of the native SNCA 3′UTR reduces toxicity of human S129A SNCA on striatal‐projecting dopamine neurons in rat substantia Nigra

Experimental models of Parkinson's disease (PD) created by aberrant expression of the alpha-synuclein (SNCA) coding region have been reported. However, noncoding regions function in normal physiology and recent in vitro studies have shown that microRNAs-7 and -153 regulate SNCA expression by binding the 3'UTR. Here, effects of different hSNCA forms were examined in vivo. Adult, male rats were injected into one substantia nigra (SN) with AAV-wtSNCA, AAV-S129A hSNCA, or AAV-S129D hSNCA either with or without a portion of the native 3'UTR. DA neurons in SN that maintained striatal (ST) projections at the end of treatment were retrogradely labeled by bilateral ST fluorogold (FG) injections and FG-positive DA neurons in SN were counted. At 5 weeks, hSNCA coding vectors reduced numbers of FG-positive neurons in injected SN compared with uninjected SN (wtSNCA, p = 0.05; S129A/D hSNCA, p = 0.01). At 7 and 9 weeks, wtSNCA- and S129D hSNCA-treated rats exhibited recovery, but S129A hSNCA-injected rats did not (p = 0.01). In contrast, numbers of FG-positive neurons were unaffected by hSNCA expression when the 3'UTR was included. When FG-positive neurons were expressed as the ratio of numbers in injected to uninjected sides, the S129A hSNCA coding vector resulted in the highest decrease at 9 weeks versus wtSNCA (p = 0.05) or S129D hSNCA (p = 0.01). Inclusion of the 3'UTR resulted in no significant differences in FG-positive neuron ratios. These data suggest that inclusion of the 3'UTR protects against S129A hSNCA-induced loss of nigrostriatal-projecting DA neurons in vivo and that mis-regulation of hSNCA expression and function at noncoding regions contribute to PD pathogenesis.

Effects of lidocaine and adrenaline combination on postoperative edema and ecchymosis in rhinoplasty

Nasal osteotomies are the most important cause of periorbital edema and ecchymosis. Injection of lidocaine and adrenaline is recommended to reduce bleeding. Whilst the lidocaine and adrenaline combination (LAC) is claimed to reduce postoperative ecchymosis and edema, this effect remains to be proven conclusively. This study, on 48 patients, was designed to investigate the effects of LAC injection on postoperative edema/ecchymosis in rhinoplasty. LAC was applied at a random side prior to the lateral osteotomy. The opposite side was used as a control. The relationship between edema/ecchymosis and the degree of LAC on the injected and uninjected sides was evaluated on the first, third and seventh day postoperatively. The relationships between edema and ecchymosis with operation time and intraoperative systolic blood pressure were also evaluated. Bleeding was reduced on the side treated with LAC (p=0.050). The degrees of edema/ecchymosis increased with increases in the duration of operation and the systolic blood pressure on the first postoperative day for the LAC-applied side (p<0.05). This correlation was not observed on the opposite side (p>0.05). Application of LAC reduces bleeding during rhinoplasty and pain control postoperatively but reduced edema and ecchymosis should not be expected following LAC application.

A rat model of Urtica ferox neuropathy

A species of stinging nettle, Urtica ferox, is indigenous to New Zealand and has caused deaths in animals and humans. We previously reported a human case of acute polyneuropathy due to U. ferox stings. We developed an experimental animal model of U. ferox toxin neuropathy to determine its neurophysiological and pathological characteristics. Male Wistar rats received either normal saline or fluid from U. ferox trichomes by injection into the epineurium of the left sciatic nerve. Neurophysiological and histological studies were carried out 5, 14 and 28 days after administration. Toxin-injected rats developed paresis of the left leg by 14 days with recovery by 28 days. Compound muscle action potentials amplitudes on the left side of toxin-administered rats at day 14 were significantly reduced compared to the right uninjected side. Toxin-injected nerves at days 5 and 14 showed a reduction in the number of myelinated fibres compared to the saline-injected nerves and frequency distributions of myelinated fibres showed a shift to smaller fibres. U. ferox neurotoxin thus produced a transient neuropathy in rat peripheral nerves with neurophysiological and pathological features suggestive of axonopathy. The identity and mechanism of action of the toxin responsible for neuropathy are uncertain.

Efficient retrograde transport of adeno-associated virus type 8 to spinal cord and dorsal root ganglion after vector delivery in muscle.

The peripheral nervous system (PNS), including peripheral nerves and dorsal root ganglion (DRG), is involved in numerous neurological disorders, such as peripheral neuropathies (diabetic neuropathy, chronic pain, etc.) and demyelination diseases (multiple sclerosis, congenital muscular dystrophy, Charcot-Marie-Tooth disease, etc.). Effective clinical interventions for those diseases are very limited. Gene therapy represents a novel therapeutic strategy for the PNS diseases, especially with simply and minimally invasive delivery methods. Previously, we have shown that adeno-associated virus type 8 (AAV8) can efficiently transduce muscles body wide by a simple intraperitoneal injection in neonatal mice. In this study, we investigated the capacity of AAV8 in transducing PNS in neonatal mice by intraperitoneal injection and also in adult mice by intramuscular injection. Efficient and long-term gene transfer was found in the white matter of the spinal cord, DRG neurons, and peripheral nerves in both groups, treated either as neonates or as adults, particularly neonates. In the adult mice injected with AAV8 in tibialis anterior and gastrocnemius muscles in one of the hind legs, more neurons were transduced in the lower part of the spinal cord than in the upper part; the DRG neurons were transduced more on the vector-injected side than in the contralateral uninjected side. Few cells in the gray matter of the spinal cord were transduced regardless of the delivery methods and age of the mice. These results support the mechanism of vector retrograde transport and suggest that AAV8 crosses blood-nerve barrier poorly. Our finding should have important implications in gene therapy for peripheral neurological disorders.

P27BBP/eIF6 acts as an anti-apoptotic factor upstream of Bcl-2 during Xenopus laevis development

p27(BBP)/eIF6 (beta4-binding protein/eukaryotic initiation factor 6) regulates the joining of 40S and 60S ribosomal subunits, on receptor for activated C kinase 1 binding and protein kinase C phosphorylation in serine 235. In Xenopus, p27(BBP)/eIF6 is abundantly expressed in the majority of the embryonic anlagen. Although p27(BBP)/eIF6 abundance may be required for a general regulation of protein synthesis, our data suggest that p27(BBP)/eIF6 may target the translation of specific mRNAs. We injected Xp27(BBP)/eIF6 mRNA in one blastomere of two-cell-stage embryos and obtained a bent phenotype, the curvature being lateral with respect to the embryo antero-posterior axis. The injected side had fewer apoptotic cells than the uninjected side, whereas cell proliferation appeared unaffected. Accordingly, in Xp27(BBP)/eIF6 morphants, endogenous apoptosis increased. Injection of Xp27(BBP)/eIF6 point mutants indicated that the anti-apoptotic action of Xp27(BBP)/eIF6 requires the conserved S235. The bent phenotype was also obtained with B-cell lymphoma gene-2 (Bcl-2) overexpression and was rescued by Bcl-2-associated X protein (Bax)/Xp27(BBP)/eIF6 co-injection. In addition, embryos overexpressing Xp27(BBP)/eIF6 had a higher amount of Bcl-2 and an unchanged amount of Bax with respect to controls. In Xp27(BBP)/eIF6 morphants, Bcl-2 levels were unaffected and Bax levels were higher than in the controls. Thus, we propose that Xp27(BBP)/eIF6 is part of a mechanism acting on the specific translation of messengers regulating cell survival. In particular, we suggest that Xp27(BBP)/eIF6 may regulate the translation of factors upstream of Bcl-2/Bax.

Long-term Transgene Expression in the Central Nervous System Using DNA Nanoparticles

This study demonstrates proof of concept for delivery and expression of compacted plasmid DNA in the central nervous system. Plasmid DNA was compacted with polyethylene glycol substituted lysine 30-mer peptides, forming rod-like nanoparticles with diameters between 8 and 11 nm. Here we show that an intracerebral injection of compacted DNA can transfect both neurons and glia, and can produce transgene expression in the striatum for up to 8 weeks, which was at least 100-fold greater than intracerebral injections of naked DNA plasmids. Bioluminescent imaging (BLI) of injected animals at the 11th postinjection week revealed significantly higher transgene activity in animals receiving compacted DNA plasmids when compared to animals receiving naked DNA. There was minimal evidence of brain inflammation. Intrastriatal injections of a compacted plasmid encoding for glial cell line-derived neurotrophic factor (pGDNF) resulted in a significant overexpression of GDNF protein in the striatum 1-3 weeks after injection.

HSP70 and Constitutively Active HSF1 Mediate Protection Against CDCrel-1-mediated Toxicity

Defects in cellular quality control mechanisms are thought to contribute to the neuropathology of Parkinson's disease (PD). Overexpressing heat shock proteins (HSPs) may constitute a powerful therapeutic strategy for PD, because they boost the ability of the cell to eliminate unwanted proteins. We investigated the neuroprotective potential of HSP70, HSP40, and H-BH, a constitutively active form of heat shock factor 1, in a rat model of PD based on adeno-associated virus (AAV) vector-mediated overexpression of CDCrel-1, a parkin substrate known to be toxic to dopaminergic neurons. AAV vector-mediated overexpression of H-BH and of HSP70 afforded similar levels of protection against CDCrel-1 toxicity, with approximately 20% improvement in survival of dopaminergic neurons as compared to the controls. The assessment of protection conferred was made using tyrosine hydroxylase (TH) and HuC/D immunohistochemistry and Fluoro-Gold retrograde tracing, and by observing the extent of preservation of spontaneous function and also the extent of drug-induced motor function. In contrast to H-BH and HSP70, HSP40 overexpression exacerbated CDCrel-1-mediated cell death. Real-time reverse transcriptase (RT)-PCR analysis showed that H-BH had the effect of upregulating endogenous HSP70 and HSP40 mRNA levels 10-fold and 4-fold over basal levels, respectively, whereas AAV vector-mediated HSP70 and HSP40 mRNA levels were over 100-fold higher. Our results suggest that a comparatively modest upregulation of multiple HSPs may be an effective approach for achieving significant neuroprotection in PD.

Successful Compensation for Dystrophin Deficiency by a Helper-dependent Adenovirus Expressing Full-length Utrophin

Helper-dependent adenovirus vector (AdV)-mediated full-length dystrophin expression leads to significant mitigation of the dystrophic phenotype of the mdx mouse. However, dystrophin, as a neoantigen, elicits antibody formation. As an alternative approach, we evaluated gene transfer of full-length murine utrophin, a functional homologue of dystrophin that is normally present only at the neuromuscular junction. A single injection in the tibialis anterior (TA) muscle of the helper-dependent adenovirus vector encoding utrophin provided very good transduction, with 58% of fibers demonstrating sarcolemmal utrophin expression in the neonates, and 35% utrophin-positive (Utr(+)) fibers in adults. The presence of utrophin prevented extensive necrosis in the neonates, halted further necrosis in the adults, and led to restoration of sarcolemmal expression of dystrophin-associated proteins up to 1 year after injection. Marked physiological improvement was observed in both neonates and adults. Neither increased humoral responses nor cellular immune responses were evident. However, there was a time-related decline of the initial high utrophin expression. Although viral DNA persisted in animals that were injected in the neonatal stage, viral DNA levels decreased in muscles of adult mice. These results demonstrate that although utrophin gene transfer leads to amelioration of the dystrophic phenotype, the effects are not sustained upon loss of utrophin expression.

Striatal delivery of CERE‐120, an AAV2 vector encoding human neurturin, enhances activity of the dopaminergic nigrostriatal system in aged monkeys

Neurturin (NTN) is a potent survival factor for midbrain dopaminergic neurons. CERE-120, an adeno-associated virus type 2 (AAV2) vector encoding human NTN (AAV2-NTN), is currently being developed as a potential therapy for Parkinson's disease. This study examined the bioactivity and safety/tolerability of AAV2-NTN in the aged monkey model of nigrostriatal dopamine insufficiency. Aged rhesus monkeys received unilateral injections of AAV2-NTN into the caudate and putamen, with each animal therefore serving as its own control. Robust expression of NTN within the nigrostriatal system was observed 8 months postadministration. (18)F-fluorodopa imaging using positron emission tomography revealed statistically significant increases in (18)F-fluorodopa uptake in the injected striatum compared with the uninjected side at 4 and 8 months. In addition, at 8 months postadministration, a significant enhancement in tyrosine hydroxylase immunoreactive fibers and an increase in the number of tyrosine hydroxylase immunoreactive cells was observed in the AAV2-NTN injected striatum compared with the uninjected side. Robust activation of phosphorylated extracellular signal-regulated kinase immunoreactivity in the substantia nigra was also observed. Histopathological analyses revealed no adverse effects of AAV2-NTN in the brain. Collectively, these results are consistent with the neurotrophic effects of NTN on the dopaminergic nigrostriatal system and extend the growing body of evidence supporting the concept that AAV2-NTN may have therapeutic benefit for Parkinson's disease.

Transient Synaptic Silencing of Developing Striate Cortex Has Persistent Effects on Visual Function and Plasticity

Neural circuits in the cerebral cortex are shaped by experience during "critical periods" early in life. For example, visual cortex is immature at the time of eye opening and gradually develops its functional properties during a sensitive period. Very few reports have addressed the role of intrinsic neural activity in cortical maturation. Here we have exploited the bacterial enzyme botulinum neurotoxin E (BoNT/E) to produce a unilateral, reversible blockade of neural activity in rat visual cortex during the sensitive period. BoNT/E is a highly selective protease that interferes with transmitter release via cleavage of the synaptic protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Unilateral, intracortical injections of BoNT/E were made at the time of eye opening and resulted in the silencing of the treated, but not contralateral, hemisphere for a period of 2 weeks. We found that visual acuity was permanently reduced in the blocked hemisphere, and the critical period for ocular dominance plasticity persisted into adulthood. Unexpectedly, these effects extended equally to the contralateral, uninjected side, demonstrating a fundamental role for interhemispheric connections in cortical maturation.

896. Long Term Transgene Expression in the Brain Delivered by SV40-Derived Vectors

Top of pageAbstract Gene transfer to the central nervous system (CNS) has been studied using various vectors. Recombinant, Tag-deleted SV40 vectors provide long-term transgene expression in vitro and in vivo in multiple non-CNS cell types, and can be used in vitro to transduce both NT2-derived and primary human neurons, and primary human brain microglia. We previously reported administering a rSV40 vector into the caudate-putamen (CP) of the rat brain using stereotaxic conditions. This vector carries HIV-1 Nef protein with a carboxyl-terminal FLAG epitope tag driven with a CMV-IEP promoter, SV(Nef-FLAG). Transgene expression was assessed 4, 7 or 14 days later by immunostaining of serial coronal cryostat sections. Numerous transgene-positive cells were seen at those times, at least several mm longitudinally away from the injection site. The present study aimed to determine whether these vectors could also deliver long term expression of a transgene, up to 3 months after unilateral injection into the CP, hippocampus, and cerebellum (lobe 6). The uninjected side, or saline injection, were used as controls. No cells on the uninjected side were immunopositive for FLAG.Sections incubated with non-immune primary antibody (immunostain negative controls) were also negative. The number of transgene-positive cells counted 1, 2 and 3 months after injection of the vector in the CP was higher than the one at d14, and showed a wider anatomic distribution. Transgene-positive cells were seen not only in the CP, but also in different brain structures along the needle track. The longitudinal extent of the transduced area was similar along the whole CP for time points greater than 4 days. In the CP, many transgene-expressing cells stained positively for NeuN, or MAP2, indicating a substantial neuronal transduction, with more than 95% of the transduced cells co-immunostaining with these neuronal markers. Astrocytes generally did not express the transgene. Long term transgene expression was also seen in the hippocampus (dentate gyrus) and in the cerebellum. More transgene-positive cells were observed when 10 microl of vector were given than with an injection of 1 microl. In the cerebellum, transgene-positive cells colocalized with calbindin, a marker for Purkinje cells. Previous studies of rSV40 gene delivery did not identify inflammatory or immunologic responses to the vector. In these studies, we also looked for evidence of inflammatory or immune reaction to the transduction process, using sections stained with neutral red (NR): no inflammation was seen at any time point in brains injected in any of the different structures. Likewise, no CD8-positive T cells were identified in sections of brains injected with SV(Nef-FLAG). These results show that SV40-derived vectors can be used to achieve long term and efficient CNS gene transfer without evidence of inflammatory or immune reaction, preferentially to neurons and to a lesser extent, microglial cells, when injected in different parenchymal structures of the rat brain.

Hsp70 Gene Transfer by Adeno-associated Virus Inhibits MPTP-Induced Nigrostriatal Degeneration in the Mouse Model of Parkinson Disease

Mitochondrial dysfunction and oxidative stress have been implicated in Parkinson disease (PD). In addition, genetic evidence points to an important role of protein misfolding, aggregation, and failure in the proteasomal degradation of specific neuronal proteins in the pathogenesis of PD. The chaperone heat-shock protein 70 (Hsp70) reduces protein misfolding and aggregation and protects cells against a variety of adverse conditions, including oxidative stress. Moreover, Hsp70 exerts antiapoptotic activity by blocking the function of several key proapoptotic factors. Recently, Hsp70 was shown to inhibit alpha-synuclein toxicity in a Drosophila model of inherited PD. Here we tested the potential of Hsp70 (approved gene symbol HSPA1A) for gene therapy in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of idiopathic PD. We show that Hsp70 gene transfer to dopamine neurons by a recombinant adeno-associated virus significantly protects the mouse dopaminergic system against MPTP-induced dopamine neuron loss and the associated decline in striatal dopamine levels and tyrosine hydroxylase-positive fibers. Hsp70 reduced MPTP-induced apoptosis in the substantia nigra, and unilateral protection of the dopaminergic system by Hsp70 was associated with increased amphetamine-induced turning toward the uninjected side. Collectively, these results suggest that increasing chaperone activity may be beneficial for the treatment of idiopathic PD.

Disinhibition of lower midbrain neurons enhances non-shivering thermogenesis in anesthetized rats

The hypothesis that the lower midbrain, specifically in and around the retrorubral field (RRF) and/or rubrospinal tract (rs), contains a tonic inhibitory mechanism on non-shivering thermogenesis (NST) was examined in urethane-anesthetized rats. It has been proposed that removal of the tonic inhibitory mechanism in the lower midbrain causes body temperature increase through disinhibition-induced activation of the central sympathetic nervous system. The present experiments were carried out to examine whether and where the proposed midbrain region contains cell bodies that tonically inhibit the NST, and if so, whether they receive any influence from the hypothalamus. Male Wistar rats were anesthetized with urethane (1.0–1.2 g/kg, i.p.), and various agents were microinjected into the RRF and rs areas of one side before and after knife-cut in the other side of the lower midbrain or isolation of the hypothalamus from the midbrain. Changes in interscapular brown adipose tissue (IBAT), rectum, and tail skin temperatures were monitored. Results: (1) unilateral midbrain procaine increased IBAT and rectal temperatures only after un-injected side of the midbrain had been pre-transected. (2) Effective midbrain sites for procaine to increase IBAT and rectal temperatures was laterally extended. (3) Tetrodotoxin microinjected into the midbrain site where procaine increased IBAT and rectal temperatures also raised both temperatures. (4) l-glutamate decreased IBAT and rectal temperatures when microinjected into one of the most inner midbrain area of procaine-sensitive sites without affecting tail skin temperature. (5) Isolation of the hypothalamus from the lower midbrain did not affect midbrain procaine-induced IBAT and rectal temperature increases. These results suggest that neurons that tonically inhibit the NST are located in the area close to the midline adjacent to the RRF and rs, and that the integrity of the neurons to tonically inhibit the NST is not affected by disconnecting the hypothalamus from the midbrain.