Severe coenurosis caused by larvae of Taenia serialis in an olive baboon (Papio anubis) in Benin.

In March 2017, a captive male juvenile (ca. 6 months old) olive baboon (Papio anubis) was brought to a primate rescue center in Benin with multiple subcutaneous swellings of unknown aetiology. At the general inspection of the body, around 15 partially mobile masses of variable sizes were found in different locations across the body. Following two surgical procedures, several cyst-like structures were removed and placed either in 10% formalin or in absolute ethanol. The cysts had a typical coenurus-like morphology. Genomic DNA was extracted from one cyst using a commercially available kit. The molecular characterization was performed by PCR amplification and sequencing of a region of the nuclear ITS-2 rDNA and a fragment of the mitochondrial 12S rDNA gene, revealing its identity as T. serialis, with 88%-98% similarity to T. serialis isolates from USA, Japan and Germany This study reports a new host for the larval form of T. serialis and confirms the role of primates as intermediate host, highlighting also the risk for human infection. Further genetic studies from multiple geographic isolates are needed to clarify the taxonomic status of this group.

A multi-centre randomized controlled trial of open vs closed management of the rectal defect after transanal endoscopic microsurgery.

AIM: Transanal endoscopic microsurgery (TEM) is a technically challenging strategy that allows expanded indications for local excision of rectal lesions. Transluminal suturing is difficult, so open management of the resultant defect is appealing. Expert opinion suggests there is more pain when the defect is left open. The aim of this study was to determine if closure of the defect created during full thickness excision of rectal lesions with TEM leads to less postoperative pain compared to leaving the defect open. METHOD: At the time of surgery, patients undergoing a full thickness TEM were randomized to sutured (TEM-S) or open (TEM-O) management of the rectal defect. At five Canadian academic colorectal surgery centres, experienced TEM surgeons enrolled patients ≥ 18 years treated by full thickness TEM. The primary outcome was postoperative pain measured by the visual analogue scale. Secondary outcomes included postoperative pain medication use and 30-day postoperative complications, including bleeding, infection and hospital readmission. RESULTS: Between March 2012 and October 2013, 50 patients were enrolled and randomized to sutured (TEM-S, n = 28) or open (TEM-O, n = 22) management of the rectal defect. There was no difference between the two study groups in postoperative pain on postoperative day 1 (2.8 vs 2.6, P = 0.76), day 3 (2.8 vs 2.1, P = 0.23) and day 7 (2.8 vs 1.7, P = 0.10). CONCLUSION: In this multicentre randomized controlled trial, there was no difference in postoperative pain between sutured or open defect management in patients having a full thickness excision with TEM.

The Noble plication: new indication for a historical procedure.

Complications following ileal pouch-anal anastomosis (IPAA) have been well-described in the literature. While rare, small bowel volvulus following IPAA has been described. We describe the successful use of the modified Noble plication in such a patient with small bowel volvulus about an elongated mesentery. This largely 'historical' technique is well-suited to manage small bowel volvulus, especially when non-resectional management is preferred.

Characterization of heterotopic cell clusters in the hippocampus of rats exposed to methylazoxymethanol in utero.

Cortical disorganization represents one of the major clinical findings in many children with medically intractable epilepsy. To study the relationship between seizure propensity and abnormal cortical structure, we have begun to characterize an animal model exhibiting aberrant neuronal clusters (heterotopia) and disruption of cortical lamination. In this model, exposing rats in utero to the DNA methylating agent methylazoxymethanol acetate (MAM; embryonic day 15) disrupts the sequence of normal brain development. In MAM-exposed rats, cells in hippocampal heterotopia exhibit neuronal morphology and do not stain with immunohistochemical markers for glia. In hippocampal slices from MAM-exposed animals, extracellular field recordings within heterotopia suggest that these dysplastic cell clusters make synaptic connections locally (i.e. within the CA1 hippocampal subregion) and also make aberrant synaptic contact with neocortical cells. Slice perfusion with bicuculline or 4-aminopyridine leads to epileptiform activity in dysplastic cell clusters that can occur independent of input from CA3. Taken together, our findings suggest that neurons within regions of abnormal hippocampal organization are capable of independent epileptiform activity generation, and can project abnormal discharge to a broad area of neocortex, as well as hippocampus.

Chloride-cotransport blockade desynchronizes neuronal discharge in the "epileptic" hippocampal slice.

Antagonism of the chloride-cotransport system in hippocampal slices has been shown to block spontaneous epileptiform (i.e., hypersynchronized) discharges without diminishing excitatory synaptic transmission. Here we test the hypotheses that chloride-cotransport blockade, with furosemide or low-chloride (low-[Cl(-)](o)) medium, desynchronizes the firing activity of neuronal populations and that this desynchronization is mediated through nonsynaptic mechanisms. Spontaneous epileptiform discharges were recorded from the CA1 and CA3 cell body layers of hippocampal slices. Treatment with low-[Cl(-)](o) medium led to cessation of spontaneous synchronized bursting in CA1 >/=5-10 min before its disappearance from CA3. During the time that CA3 continued to burst spontaneously but CA1 was silent, electrical stimulation of the Schaffer collaterals showed that hyperexcited CA1 synaptic responses were maintained. Paired intracellular recordings from CA1 pyramidal cells showed that during low-[Cl(-)](o) treatment, the timing of action potential discharges became desynchronized; desynchronization was identified with phase lags in firing times of action potentials between pairs of neurons as well as a with a broadening and diminution of the CA1 field amplitude. Continued exposure to low-[Cl(-)](o) medium increased the degree of the firing-time phase shifts between pairs of CA1 pyramidal cells until the epileptiform CA1 field potential was abolished completely. Intracellular recordings during 4-aminopyridine (4-AP) treatment showed that prolonged low-[Cl(-)](o) exposure did not diminish the frequency or amplitude of spontaneous postsynaptic potentials. CA3 antidromic responses to Schaffer collateral stimulation were not significantly affected by prolonged low-[Cl(-)](o) exposure. In contrast to CA1, paired intracellular recordings from CA3 pyramidal cells showed that chloride-cotransport blockade did not cause a significant desynchronization of action potential firing times in the CA3 subregion at the time that CA1 synchronous discharge was blocked but did reduce the number of action potentials associated with CA3 burst discharges. These data support our hypothesis that the anti-epileptic effects of chloride-cotransport antagonism in CA1 are mediated through the desynchronization of population activity. We hypothesize that interference with Na(+),K(+),2Cl(-) cotransport results in an increase in extracellular potassium ([K(+)](o)) that reduces the number of action potentials that are able to invade axonal arborizations and varicosities in all hippocampal subregions. This reduced efficacy of presynaptic action potential propagation ultimately leads to a reduction of synaptic drive and a desynchronization of the firing of CA1 pyramidal cells.

Extracellular chloride and the maintenance of spontaneous epileptiform activity in rat hippocampal slices.

Previous studies showed that furosemide blocks spontaneous epileptiform activity without diminishing synaptic transmission or reducing hyperexcited field responses to electrical stimuli. We now test the hypothesis that the antiepileptic effects of furosemide are mediated through its blockade of the Na+,K+,2Cl- cotransporter and thus should be mimicked by a reduction of extracellular chloride ([Cl-]o). In the first set of experiments, field recordings from the CA1 cell body layer of hippocampal slices showed that spontaneous bursting developed within 10-20 min in slices perfused with low-[Cl-]o (7 mM) medium but that this spontaneous epileptiform activity ceased after a further 10-20 min. Intracellular recordings from CA1 pyramidal cells showed that normal action potential discharge could be elicited by membrane depolarization, even after the tissue was perfused with low-[Cl-]o medium for >2 h. In a second set of experiments, spontaneous bursting activity was induced in slices by perfusion with high-[K+]o (10 mM), bicuculline (100 microM), or 4-aminopyridine (100 microM). In each case, recordings from the CA1 region showed that reduction of [Cl-]o to 21 mM reversibly blocked the bursting within 1 h. Similar to previous observations with furosemide treatment, low-[Cl-]o medium blocked spontaneous hypersynchronous discharges without reducing synaptic hyperexcitability (i.e., hyperexcitable field responses evoked by electrical stimulation). In a third set of experiments, prolonged exposure (>1 h after spontaneous bursting ceased) of slices to systematically varied [Cl-]o and [K+]o resulted in one of three types of events: 1) spontaneous, long-lasting, and repetitive negative field potential shifts (7 mM [Cl-]o; 3 mM [K+]o); 2) oscillations consisting of 5- to 10-mV negative shifts in the field potential, with a period of approximately 1 cycle/40 s (16 mM [Cl-]o; 12 mM [K+]o); and 3) shorter, infrequently occurring negative field shifts lasting 20-40 s (21 mM [Cl-]o; 3 mM [K+]o). Our observations indicate that the effects of low [Cl-]o on neuronal synchronization and spontaneous discharge are time dependent. Similar effects were seen with furosemide and low [Cl-]o, consistent with the hypothesis that the antiepileptic effect of furosemide is mediated by the drug's effect on chloride transporters. Finally, the results of altering extracellular potassium along with chloride suggest that blockade of the Na+, K+,2Cl- cotransporter, which normally transports chloride from the extracellular space into glial cells, is key to these antiepileptic effects.

Osmolarity, ionic flux, and changes in brain excitability.

The majority of modern epilepsy research has focused on possible abnormalities in synaptic and intrinsic neuronal properties--as likely epileptogenic mechanisms as well as the targets for developing novel antiepileptic treatments. However, many other processes in the central nervous system contribute to neuronal excitability and synchronization. Regulation of ionic balance is one such set of critical processes, involving a complex array of molecules for moving ions into and out of brain cells--both neurons and glia. Alterations in extracellular-to-intracellular ion gradients can have both direct and indirect effects on neuronal discharge. We have found, for example, that when hippocampal slices are exposed to hypo-osmotic bathing medium, the cells not only swell, but there is also a significant increase in the amplitude of a delayed rectifier potassium current in inhibitory interneurons--an effect that may contribute to the increase in tissue excitability associated with hypo-osmolar treatments. In contrast, antagonists of the chloride co-transporter, furosemide or bumetanide, block epileptiform activity in both in vitro and in vivo preparations. This antiepileptic effect is presumably due to the drugs' ability to block chloride co-transport. Indeed, prolonged tissue exposure to low levels of extracellular chloride have a parallel action. These experiments indicate that manipulation of ionic balance may not only facilitate epileptiform activities, but may also provide insight into new therapeutic strategies to block seizures.

Intrinsic optical changes in neuronal tissue. Basic mechanisms.

Associated with changes in the level of physiological activity in neuronal tissue are changes in the intrinsic optical properties of the tissue. As a consequence, it is possible to optically monitor neuronal activity without the use of dyes or other contrast-enhancing agents. Such optical techniques have been applied in the laboratory for more than 50 years. Recent developments in near-infrared spectroscopy and intraoperative optical imaging have suggested a number of clinically important applications of this technology. This article provides an overview of what is known about the physiological correlates and underlying mechanisms associated with activity-evoked optical changes in neuronal tissue.

Evidence for p53-mediated modulation of neuronal viability.

A role for p53-related modulation of neuronal viability has been suggested by the finding that p53 expression is increased in damaged neurons in models of ischemia and epilepsy. These findings were recently extended with the demonstration that mice deficient in p53 ("knock-out" mice) exhibit almost complete protection from seizure-induced brain injury, whereas wild-type mice display significant neuronal cell loss in the hippocampus and other brain regions. Because the p53 knock-out mice used in the latter study expressed a global p53 deficiency in all cell types, it was not possible to conclude that protection was conferred by the exclusive absence of p53 in neurons. Therefore, in the present study, we determined whether p53 expression in isolated neurons is directly coupled to a loss of viability associated with excitotoxic challenge. Primary cultures of hippocampal or cortical neurons were derived from animals containing p53 (+/+, +/-) or those deficient in p53 (-/-). p53-Deficient neurons appeared identical to wild-type neurons with respect to morphology, neurofilament expression, and resting levels of intracellular calcium. Neurons containing at least one copy of p53 were severely damaged by exposure to kainic acid or glutamate. Cell damage was assessed by direct cell counting and by nuclear morphology after propidium iodide staining of DNA. In contrast, neurons deficient in p53 (-/-) exhibited little or no damage in response to excitotoxin treatment. Despite their divergent outcomes, p53 (+/+) and p53 (-/-) neurons demonstrated similar sustained elevations in intracellular calcium levels triggered by glutamate exposure. Restoring p53 expression to p53-deficient neurons, using adenovirus-mediated transduction, was sufficient to promote neuronal cell death even in the absence of excitotoxin. These results demonstrate a direct relationship between p53 expression and loss of viability in CNS neurons.

Multiple genotype analysis and sexing of IVF bovine embryos.

Twenty-one in vitro-fertilized bovine blastocysts were quartered, lysed and subjected to primer elongation preamplification (PEP) procedure, allowing for the analysis of up to 40 genotypes per quarter embryo. The quarter-embryos were sexed by polymerase chain reaction (PCR) using BRY.1, Bov97M and ZFX/ZFY loci, and then genotyped for k-casein, bovine leukocyte adhesion deficiency (BLAD) and microsatellite D9S1. The mitochondrial cytochrome B locus was used as an internal control with a 95% success rate. The PEP procedure amplified genomic fragments in 93% of all cases. The embryos were identified to be 11 males and 10 females. Sexing accuracy was 87% for BRY.1, 97% for ZFX/ZFY and 100% for Bov97M. False genotyping was due mostly to amplification of BRY.1 in the female embryos and to the nonamplification of the ZFY locus in the male embryos. The results indicate that the combined use of Bov97M and ZFX/ZFY loci is a highly accurate procedure for sexing bovine embryos. Genotyping for kappa-casein, D9S1 and BLAD was successful in 94, 99 and 91% of assays, respectively. Sex ratios and allele frequencies of embryos for gk-casein, BLAD and D9S1 were all close to the observed frequencies in the Israeli Holstein population. These results support the conclusion that the genotyping of embryos is as accurate as that of mature animals. Thus, marker-assisted selection can be efficiently applied at the preimplantation embryo level for loci of economic importance.

Enhanced optical imaging of human gliomas and tumor margins.

One of the potential variables affecting the overall survival and quality of life of patients with intracranial gliomas is the extent of tumor resection that results in the smallest volume of residual disease. A technique involving enhanced optical imaging of human gliomas has the potential to localize tumors, identify tumor remaining at the resection margins, and determine the grade of the tumor. In a preliminary study involving nine patients undergoing surgery for the removal of intrinsic brain tumors, enhanced optical imaging was performed using indocyanine green as an intravenous contrast-enhancement agent. Optical images were obtained before and after injection of the indocyanine green. The studies in the nine patients showed differences in the dynamic optical signals among normal brain, low-grade astrocytomas, and malignant astrocytomas. Optical imaging of the resection margins in malignant tumors showed differences between adjacent normal tissue and remaining tumor tissue. Enhanced optical imaging of human gliomas using a contrast-enhancing dye, indocyanine green, provides a potential means to differentiate between normal brain and tumor tissue at the cortical surface and the depths of the resection margins. Having the ability to obtain real-time information and feedback in the operating room may allow neurosurgeons to maximize the extent of tumor resection while sparing normal brain and increasing the diagnostic accuracy of intraoperative biopsies. Enhanced optical imaging potentially could facilitate the accuracy and safety of surgery when tumors are removed at sites even outside the central nervous system.

Dissociation of synchronization and excitability in furosemide blockade of epileptiform activity.

Furosemide, a chloride cotransport inhibitor, reversibly blocked synchronized burst discharges in hippocampal slices without reducing the pyramidal cell response to single electrical stimuli. Images of the intrinsic optical signal acquired during these slice experiments indicated that furosemide coincidentally blocked changes in extracellular space. In urethane-anesthetized rats, systemically injected furosemide blocked kainic acid-induced electrical discharges recorded from cortex. These results suggest that (i) neuronal synchronization involved in epileptiform activity can be dissociated from synaptic excitability; (ii) nonsynaptic mechanisms, possibly associated with furosemide-sensitive cell volume regulation, may be critical for synchronization of neuronal activity; and (iii) agents that affect extracellular volume may have clinical utility as antiepileptic drugs.

Enhanced optical imaging of rat gliomas and tumor margins.

Current intraoperative methods used to maximize the extent of tumor removal are limited to intraoperative biopsies, ultrasound, and stereotactic volumetric resections. A new technique involving the optical imaging of an intravenously injected dye has the potential to localize tumors and their margins with a high degree of accuracy. In a rat glioma model, enhanced optical imaging was performed and indocyanine green was used as the contrast-enhancing agent. In all 22 animals, the peak optical change in the tumor was greater than in the ipsilateral brain around the tumor and the contralateral normal hemisphere. The clearance of the dye was significantly delayed to a greater extent in the tumor than in the brain around the tumor and the normal brain. After attempts were made at complete microscopic resection, enhanced optical imaging of the tumor margins and the histological samples demonstrated a specificity of 93% and a sensitivity of 89.5%. Enhanced optical imaging was capable of outlining the tumor even when the imaging was done through the cranium. The optical imaging of rat gliomas with a contrast-enhancing dye is able to differentiate between normal brain and tumor tissue both at the cortical surface and at the tumor margins. The application of these studies in an intraoperative clinical setting may allow for the more accurate determination of tumor margins and may increase the extent of tumor removal.

Repeated NMDA receptor activation induces distinct intracellular calcium changes in subpopulations of striatal neurons in vitro.

The mechanisms underlying long-term calcium changes evoked by excitatory amino acids have not been previously examined in striatal neurons. Fura-2 fluorescence measurements were used to examine intracellular calcium concentration ([Ca2+]i) changes due to repeated N-methyl-D-aspartate (NMDA) receptor activation, in primary cultures of murine striatal neurons. Three applications of 200 microM NMDA (for 2 min, each application separated by 7 min), in 0 magnesium-containing artificial cerebral spinal fluid, elicited three distinct responses. In 50 +/- 8% of the NMDA-responsive neurons, no persistent increases in [Ca2+]i (final [Ca2+]i < or = 150% baseline) were observed, while in 33 +/- 7% and 17 +/- 3% of the cells, sustained (peak response > final [Ca2+]i > 150% baseline) and uncontrolled increases (final [Ca2+]i > or = peak response) were observed, respectively. NMDA-responsive neurons that were intensely immunoreactive for the calcium binding protein calbindin-D28k never exhibited uncontrolled increases in [Ca2+]i. Removal of extracellular Ca2+ significantly attenuated sustained, but not uncontrolled, increases in [Ca2+]i; sustained increases in some neurons were also attenuated by application of verapamil (100 microM) or MK-801 (1 microM). Pre-treatment of striatal neurons with the protein kinase C blocker sphingosine (20 microM), virtually eliminated the development of sustained or uncontrolled increases in [Ca2+]i. These findings suggest that specific intracellular mechanisms regulate the distinct [Ca2+]i responses of subpopulations of striatal neurons to repeated NMDA receptor activation.

Optical imaging of epileptiform and functional activity in human cerebral cortex.

Optical imaging of animal somatosensory, olfactory and visual cortices has revealed maps of functional activity. In non-human primates, high-resolution maps of the visual cortex have been obtained using only an intrinsic reflection signal. Although the time course of the signal is slower than membrane potential changes, the maximum optical changes correspond to the maximal neuronal activity. The intrinsic optical signal may represent the flow of ionic currents, oxygen delivery, changes in blood volume, potassium accumulation or glial swelling. Here we use similar techniques to obtain maps from human cortex during stimulation-evoked epileptiform afterdischarges and cognitively evoked functional activity. Optical changes increased in magnitude as the intensity and duration of the afterdischarges increased. In areas surrounding the afterdischarge activity, optical changes were in the opposite direction and possibly represent an inhibitory surround. Large optical changes were found in the sensory cortex during tongue movement and in Broca's and Wernicke's language areas during naming exercises. The adaptation of high-resolution optical imaging for use on human cortex provides a new technique for investigation of the organization of the sensory and motor cortices, language, and other cognitive processes.

Imaging of synaptically evoked intrinsic optical signals in hippocampal slices.

Imaging analysis techniques were used to examine changes in the intrinsic optical properties in hippocampal brain slices that occurred during synaptic activity evoked by Schaffer collateral stimulation in CA1. Repetitive synaptic activity was associated with an increase in light transmission in the synaptic region in stratum radiatum. The effect was seen at wavelengths of light between 450 and 800 nm but was of greater amplitude at longer wavelengths. Blocking synaptic transmission with either Ca(2+)-free EGTA perfusate or kynurenic acid (an excitatory amino acid antagonist) blocked the optical signal, indicating that it resulted from postsynaptic activation of the cells and was not due to presynaptic fiber volleys or transmitter release alone. Because the optical changes were blocked by reducing extracellular Cl- (by replacement with gluconate) or by furosemide (an anion transport inhibitor), increased Cl- transport (conceivably Na-K-2Cl cotransport) may generate these signals possibly by causing cellular swelling and thereby less light scattering. These optical changes were not blocked, however, by bicarbonate-free solution, indicating that bicarbonate transport may not be involved. Changes in the intrinsic optical signal could be related to glial swelling due to K+ released during neuronal activity because high-K(+)-induced swelling of cultured astrocytes is blocked by furosemide and low-Cl- solution. Intrinsic optical signals of neuronal tissue should be considered when voltage- or ion-sensitive dyes are used.

Modulation of intracellular Ca++ in cultured astrocytes by influx through voltage-activated Ca++ channels.

Fura-2 and indo-1 fluorescence measurements were used to examine intracellular Ca++ concentration ([Ca++]i) and its modulation by voltage-activated influx in murine cortical astrocytes in primary cell culture. Extracellular K+ was increased from 5 to 50 mM to depolarize cells to determine if Ca++ influx through voltage activated Ca++ channels could alter [Ca++]i. In confluent 4 to 6 weeks in vitro astrocyte cultures 50 mM K+ increased [Ca++]i 3-4-fold (from 150 nM up to 550 nM); this increase was blocked by nifedipine and enhanced by BayK 8644 indicating that influx was through L-type channels. However, in 1 to 2 weeks in vitro astrocyte cultures, high K+ reduced [Ca++]i. L-type channels were apparently present in these cells because high K+ in combination with BayK 8644 increased [Ca++]i. Following pretreatment of 1 to 2 weeks in vitro astrocytes with dibutyryl cAMP (dbcAMP) high K+ increased [Ca++]i in the absence of BayK 8644 indicating enhanced activity of Ca++ channels in agreement with previous voltage-clamp studies. Ca++ influx through voltage-activated channels in cultured cortical astrocytes can substantially increase [Ca++]i and these channels can be dynamically modulated by dihydropyridines. Immature astrocytes may express 'silent' or inactive Ca++ channels or have a much lower number of channels.