Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery.

BACKGROUND: Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice. RESULTS: Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependent manner. More than 90% of transduced cells were small and medium sized neurons (< 700 microm 2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (approximately 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell types. CONCLUSION: We have found that rAAV2/6 is an efficient vector to deliver transgenes to nociceptive neurons in mice. Furthermore, the characterization of the transduction profile may facilitate gene transfer studies to dissect mechanisms behind neuropathic pain.

Diagnostic approach to pupillary abnormalities.

PURPOSE OF REVIEW: This article presents an overview of the common and various kinds of pupillary disorders that can be encountered in an outpatient setting. RECENT FINDINGS: The dorsal midbrain is a site where lesions may produce either an afferent or an efferent pupillary defect. The classic pupillary syndrome secondary to a dorsal midbrain lesion is bilateral light-near dissociation. Another recognized deficit is bilateral mydriasis. Recent reports have documented unilateral mydriasis, unilateral light-near dissociation, and a relative afferent pupillary defect without visual loss in association with lesions of the dorsal midbrain. These are rare syndromes. SUMMARY: Careful history and examination can often identify and localize the pupillary disorder, as well as guide appropriate evaluation.

Digenetic trematodes (Cryptogonimidae, Acanthostominae) parasites of the Caiman, Caiman crocodilus yacare (Reptilia, Crocodylia) from the Pantanal Mato-grossense, Brazil, with the description of a new species

Proctocaccum dorsale n. sp. is described and Caimanicola marajoara Freitas & Lent, 1938 is redescribed based on specimens collected from Caiman crocodilus yacare (Daudin) from the Pantanal Mato-grossense, State of Mato Grosso do Sul, Brasil. This report extends south ward the known geographic distribution of C. marajoara. It is the first record of the genus Proctocaccum in South America, and in the caiman. Proctocaccum dorsale n. sp. differs from the other eight species in the genus by the dorsal location of the anal openings, instead of being lateral or in the posterior extremity of the body.

Gundlachia dutrae: n. sp. from northwest Brazil (Mollusca: Basommatophora: Ancylidae)

A new species of Gundlachia, Gundlachia dutrae is described from northwest Brazil. It is distinguishable from other congenerie species by characteristics of the shell, radula and internal organs. Shell relatively high. Aperture near-circular; periostracum dark brown without periostracal hairs. Apex slightly inclined to the right, projected but not hooked, with an apical depression surrounted by a sculpture of well-marked irregular punctations. Shell surface with prominent radial sculpture. No septate specimens were observed. Ratios (n= 59): shell width/shell lenght = 0,66- 0,79 (mean 0,73); shell height/shell length = 0,32- 0,45 ( mean 0,37); shell height/shell width = 0,43- 0,63 (mean 0,51). Body of normal ancylid type; mantle pigmentation dark brown or black, concentrated along the mantle collar. The dorsal surface of the right anterior muscle is elongated and medially constricted. The left anterior and the posterior muscles are almost elliptical. Adhesive area is V-shaped. Pseudobranch unpigmented bearing a very small and thin dorsal lobe. Ovotestis with more than 25 unbranched diverticula. Ovispermiduct with seminal vesicle rather developed. Elongated nidamental gland continous with the glandular wall of the uterus. Nidamental gland appendix ending into a bulbous swelling Spermathecal body almost rounded. Well-developed prostate with five long diverticula. Ejaculatory complex with long glandular flagellum, without a penis or true ultra-penis. "Penis sheath" developed. "Ultra-penis" projected as a tube inside the lumem of prepuce, with a slit between "ultra-penis" and "penis sheath". Rachidian tooth tetracuspid, with two median cusps assymmetrical and aculeated. Lateral teeth tricuspid, with a reduced endocon and a prominent mesocon. A well marked gap occurs between meso and ectocon. Marginal teeth similar to lateral ones. Jaw T-shaped, with about 28 dorsal plates.

Potassium restores vasorelaxation of resistance arterioles in non-hypertensive DOCA/salt fed mice.

BACKGROUND: Potassium-enriched diets exert renal and cardiovascular protective effects, but the underlying mechanisms are largely unknown. METHODS: Using the dorsal skinfold chamber model for intravital microscopy, we examined endothelium-dependent vasorelaxation of precapillary resistance arterioles in response to acetylcholine or the NO donor SNAP in awake mice. Experiments were performed in uni-nephrectomized one renin gene (Ren-1c) C57BL/6 mice (control group) and in mice having received a continuous administration of deoxycorticosterone acetate and a dietary supplementation of 1% sodium chloride for 8weeks (DOCA/salt group). An additional group of DOCA/salt treated animals received a dietary supplement of 0.4% KCl for 3weeks prior to the experiments (DOCA/salt + potassium group). RESULTS: DOCA/salt treatment for 8weeks resulted in hypokalemia, but blood pressure remained unchanged. In DOCA/salt mice, relaxation of resistance arterioles was blunted in response to acetylcholine, and to a lesser extent to SNAP, suggesting endothelial dysfunction. Endothelium-dependent vasorelaxation was restored by the potassium-enriched diet. CONCLUSION: This study is the first to demonstrate a protective effect of potassium on endothelium-dependent vasorelaxation in the absence of confounding anti-hypertensive effects, as observed in most animal models and the clinical situation. We propose that the known cardio- and nephro-protective effects of potassium might - at least in part - be mediated by the salutary effects on endothelium-dependent arteriolar relaxation.

Thyroid hormone enhances transected axonal regeneration and muscle reinnervation following rat sciatic nerve injury.

Improvement of nerve regeneration and functional recovery following nerve injury is a challenging problem in clinical research. We have already shown that following rat sciatic nerve transection, the local administration of triiodothyronine (T3) significantly increased the number and the myelination of regenerated axons. Functional recovery is a sum of the number of regenerated axons and reinnervation of denervated peripheral targets. In the present study, we investigated whether the increased number of regenerated axons by T3-treatment is linked to improved reinnervation of hind limb muscles. After transection of rat sciatic nerves, silicone or biodegradable nerve guides were implanted and filled with either T3 or phosphate buffer solution (PBS). Neuromuscular junctions (NMJs) were analyzed on gastrocnemius and plantar muscle sections stained with rhodamine alpha-bungarotoxin and neurofilament antibody. Four weeks after surgery, most end-plates (EPs) of operated limbs were still denervated and no effect of T3 on muscle reinnervation was detected at this stage of nerve repair. In contrast, after 14 weeks of nerve regeneration, T3 clearly enhanced the reinnervation of gastrocnemius and plantar EPs, demonstrated by significantly higher recovery of size and shape complexity of reinnervated EPs and also by increased acetylcholine receptor (AChRs) density on post synaptic membranes compared to PBS-treated EPs. The stimulating effect of T3 on EP reinnervation is confirmed by a higher index of compound muscle action potentials recorded in gastrocnemius muscles. In conclusion, our results provide for the first time strong evidence that T3 enhances the restoration of NMJ structure and improves synaptic transmission.

Neotropical Monogenoidea. 23. Two new species of Gyrodactylus (Gyrodactylidae) from a Cichlid and an Erythrinid fish of Southeastern Brazil

Two new species of Gyrodactylus Nordmann, 1832 (Platyhelminthes, Monogenoidea) are described from fishes collected from southeastern Brazil. Gyrodactylus geophagensis n. sp. was collected from the body surface of the "cará", Geophagus brasiliensis (Quoy and Gaimard, 1824) (Cichlidae), from the Rio da Guarda, state of Rio de Janeiro; its major diagnostic features are the morphology of the anchor with a short, truncate superficial root and the shape of the hooks - with a long, delicate shaft. Gyrodactylus trairae n. sp. parasitizes the body surface of the "traíra", Hoplias aff. malabaricus (Bloch, 1794) (Erythrinidae), from the rio Guandu, state of Rio de Janeiro and can be easily differentiated from other species of the genus by having a thin, dorsal bridge, connecting the superficial bar with the spathulated shield. These are the first species of Gyrodactylus formally reported from Brazil. Presently, 26 species of Gyrodactylidae are known from freshwater fishes in the neotropical region; a list of these species is provided.

Hermanlentia n. gen. da tribo Triatomini, com um rol de espécies de Triatominae (Hemiptera, Reduviidae)

Hermanlentia n. gen. of Tribe Triatomini, with a List of Species of Triatominae (Hemiptera, Reduviidae) - Hermanlentia n. gen. was established in the tribe Triatomini, based on differences of external morphology, phallic structures and chromatic characters of Triatoma matsunoi Fernandez-Loayza 1989, in relation to other species of Triatoma. The head is long with disproportionally small eyes 1:0,16; the relation of the head and the pronotum is 1: 0,4; and the coloration of the hemelytron is whitish. Characters of the male genitalia that distinguish the new genus are the articulatory apparatus with twice the size of the aedeagus, phallosoma with fingerlike apex, 1 + 1 dorsal conjuntive processes with the inner edge toothed, and absence of endosoma process and vesica

Morphological Aspects of the Larval Instars of Chrysomya albiceps (Diptera, Calliphoridae) Reared in the Laboratory

In order to study the morphology of young Chrysomya albiceps forms, newly hatched larvae were collected at 2 hr intervals, during the first 56 hr; after this time the collection was made at 12 hr intervals. For identification and drawing, larvae were placed between a slide and a coverslip. The cephalopharyngeal skeletons along with the first and last segments were cut off for observation of their structures and spiracles. The larvae present microspines, which are distributed randomly throughout the 12 segments of the body surface; the cephalopharyngeal skeleton varies in shape and extent of sclerotization according to larval instar; the second and third instars have relatively long processes (tubercles) on the dorsal, lateral and ventral surfaces, with microspine circles on the terminal portion

Egg Structures of Anopheles fluminensis and Anopheles shannoni

Eggs of two species belonging to the Arribalzagia Series of the Laticorn Section of Anopheles (Anopheles) collected in Brazil are described from scanning electron micrographs. The An. fluminensis egg is long with shallow floats displaced far dorsally. The narrow deck region is overlain by a frill modified into prominent ridges that are nearly continuous to both ends of the egg. Slightly opened decks at both poles contain an average of four lobed tubercles. Polygonal, plastron-type chorionic cells cover the lateral and dorsal surfaces. The egg of An. shannoni is unique in possessing 22-27 fingerlike filaments that project with regular spacing from each of its massive floats. These filaments and their bases are highly perforated and are believed to trap air and support flotation of the egg with the dorsal surface up, contrary to the usual orientation for anophelines. The eggs are compared with those of related species bearing similar structures, notably An. fluminensis with An. mediopunctatus s.s and An. shannoni with An. peryassui

Paraorientatractis semiannulata n. g., n. sp. (Cosmocercoidea: Atractidae) from the Large Intestine of the SideNecked Turtle, Podocnemis unifilis Troschel, 1848 (Testudines: Pelomedusidae) in Brazil

Specimens collected from the large intestine of the sidenecked turtle Podocnemis unifilis Troschel, 1848 in the region of Cuminá and Trombetas rivers near Pará, Brazil are assigned to a new genus and new species of the nematode superfamily Cosmocercoidea and family Atractidae and named Paraorientatractis semiannulata. The new genus is separated from the nearest genus Orientatractis by the funnelshaped mouth opening, the presence of 4 distinct lips, 4 papillae in the internal cycle, one on each lip margin, 2 lateral amphids with large amphidial pores and absence of submedian papillae. It is also separated from Orientatractis and Proatractis by the presence of striated lateral alae which curve dorsally extending from mid oesophagus to mid tail, the difference in size of the vulvar opening and the presence of large transverse ridges or semiannules on the dorsal surface. The new species can be separated from the species of the genera Orientatractis and Proatractis by the characters that distinguish the genera and the arrangement of the caudal papillae on the male. A host/parasite list for Podocnemis spp. is included

Morphological Study of Adult Male Worms of Schistosoma mansoni Sambon, 1907 by Scanning Electron Microscopy

Tubercles, spines and sensory receptors are the most studied structures of adult male worms of Schistosoma mansoni isolated in other countries. The purpose of this investigation was to properly define these structures in Brazilian worms. Specimens 7-8 weeks after infection were recovered from albino SW mice and from a wild rodent (Nectomys squamipes) and processed for scanning electron microscopy studies. Photomicrographs of the anterior region with the aspects related to the outer and inner regions of both suckers were considered. The ventral portion of the middle region was represented by the anterior of gynaecophoric canal while the dorsal surface was studied in its ventral and dorsal regions mainly focusing the aspect of the tubercles, spines and sensorial papillae. The outer surface of the oral sucker is spiny and spines are bigger, sharp with sensory receptors in their posterior edge. Tubercles with spines or receptors are more concentrated in the middle region and in one of the margins of the gynaecophoric canal. An excretory pore-like structure in the posterior portion was observed. The gynaecophoric canal has few sensory structures, spines broadned in their mid-region and are sharp pointed at the distal end. It was concluded that the presently studied characters are similar to those previously reported

Angiotensinergic neurotransmission in the peripheral autonomic nervous system.

Angiotensin (Ang) II has for long been identified as a neuropeptide located within neurons and pathways of the central nervous system involved in the control of thirst and cardio-vascular homeostasis. The presence of Ang II in ganglionic neurons of celiac, dorsal root, and trigeminal ganglia has only recently been described in humans and rats. Ang II-containing fibers were also found in the mesenteric artery and the heart, together with intrinsic Ang II-containing cardiac neurons. Ganglionic neurons express angiotensinogen and co-localize it with Ang II. Its intraneuronal production as a neuropeptide appears to involve angiotensinogen processing enzymes other than renin. Immunocytochemical and gene expression data suggest that neuronal Ang II acts as a neuromodulatory peptide and co-transmitter in the peripheral autonomic, and also sensory nervous system. Neuronal Ang II probably competes with humoral Ang II for effector cell activation. Its functional role, however, still remains to be determined. Angiotensinergic neurotransmission in the autonomic nervous system is a potential new target for therapeutic interventions in many common diseases such as essential hypertension, heart failure, and cardiac arrhythmia.

Giardia agilis: Ultrastructure of the Trophozoites in the Frog Intestine

Intestine samples of Bufo sp. tadpoles with parasitism confirmed for Giardia agilis were studied by transmission electron microscopy. The G. agilis trophozoites were long and thin. The plasma membrane was sometimes undulated and the cytoplasm, adjacent to the dorsal and ventral regions, showed numerous vacuoles. The two nuclei presented prominent nucleoli. The cytoplasm was electron-dense with free ribosomes, glycogen and rough endoplasmic reticulum-like structures. Polyhedral inclusions were observed in the cytoplasm and outside the protozoan; some of these inclusions exhibited membrane disruption. The flagella ultrastructure is typical, with the caudal pair accompanied by the funis. Next to the anterior pair, osmiophilic material was noticed. The ventro-lateral flange was short and thick, supported by the marginal plates that penetrated into its distal extremity; only its distal portion had adjacent osmiophilic filament. The G. agilis trophozoites showed the general subcellular feature of the genus. However, the ventro-lateral flange ultrastructure was an intermediate type between G. muris and G. duodenalis.

Do glial cells control pain?

Management of chronic pain is a real challenge, and current treatments focusing on blocking neurotransmission in the pain pathway have only resulted in limited success. Activation of glia cells has been widely implicated in neuroinflammation in the central nervous system, leading to neruodegeneration in many disease conditions such as Alzheimer's and multiple sclerosis. The inflammatory mediators released by activated glial cells, such as tumor necrosis factor-α and interleukin-1β can not only cause neurodegeneration in these disease conditions, but also cause abnormal pain by acting on spinal cord dorsal horn neurons in injury conditions. Pain can also be potentiated by growth factors such as BDNF and bFGF that are produced by glia to protect neurons. Thus, glia cells can powerfully control pain when they are activated to produce various pain mediators. We will review accumulating evidence supporting an important role of microglia cells in the spinal cord for pain control under injury conditions (e.g. nerve injury). We will also discuss possible signaling mechanisms in particular MAP kinase pathways that are critical for glia control of pain. Investigating signaling mechanisms in microglia may lead to more effective management of devastating chronic pain.