Mutations in ATP2C1, encoding a calcium pump, cause Hailey-Hailey disease.

Hailey-Hailey disease (HHD, MIM 16960) is inherited in an autosomal dominant manner and characterized by persistent blisters and erosions of the skin. Impaired intercellular adhesion and epidermal blistering also occur in individuals with pemphigus (which is due to autoantibodies directed against desmosomal proteins) and in patients with Darier disease (DD, MIM 124200), which is caused by mutations in a gene encoding a sarco/endoplasmic reticulum (ER)-Golgi calcium pump. We report here the identification of mutations in ATP2C1, encoding the human homologue of an ATP-powered pump that sequesters calcium into the Golgi in yeast, in 21 HHD kindreds. Regulation of cytoplasmic calcium is impaired in cultured keratinocytes from HHD patients, and the normal epidermal calcium gradient is attenuated in vivo in HHD patients. Our findings not only provide an understanding of the molecular basis of HHD, but also underscore the importance of calcium control to the functioning of stratified squamous epithelia.

A microPET study of the regional distribution of [11C]-PK11195 binding following temporary focal cerebral ischemia in the rat. Correlation with post mortem mapping of microglia activation.

BACKGROUND: Post-stroke microglial activation (MA) may have both neurotoxic and pro-repair effects, particularly in the salvaged penumbra. Mapping MA in vivo is therefore an important goal. 11C-PK11195, a ligand for the 18 kDa translocator protein, is the reference radioligand for MA imaging, but a correlation between the regional distributions of in vivo tracer binding and post mortem MA after stroke, as assessed with PET and immunohistochemistry, respectively, has not been demonstrated so far. Here we performed 11C-PK11195 microPET in a rat model previously shown to induce extensive cortical MA, and determined the correlation between 11C-PK11195 and immunostaining with the CD11 antibody OX42, so as to verify the presence of activated microglia, in a template of PET-resolution size regions-of-interest (ROIs) spanning the whole affected hemisphere. METHODS: Adult spontaneously hypertensive rats underwent 45 min distal middle cerebral artery occlusion and 11C-PK11195 PET at Days 2 and 14 after stroke according to a longitudinal design. Following perfusion-fixation at Day 14, brains were removed and coronally cut for OX42 staining. 11C-PK11195 binding potential (BPND) parametric maps were generated, and in each rat both BP(ND) and OX42 (intensity×extent score) were obtained in the same set of 44 ROIs extracted from a cytoarchitectonic atlas to cover the whole hemisphere. Correlations were computed across the 44 ROIs both within and across subjects. RESULTS: Significant BPND increases were observed in both the infarct and surrounding areas in all rats at day 14; less strong but still significant increases were present at day 2. There were highly significant (all p<0.001) positive correlations, both within- and across-subjects, between day 14 BPND values and OX42 scores. CONCLUSIONS: The correlation between Day 14 11C-PK11195 and OX42 across the affected hemisphere from the same brain regions and animals further supports the validity of 11C-PK11195 as an in vivo imaging marker of MA following stroke. The finding of statistically significant increases in 11C-PK11195 as early as 48 h after stroke is novel. These results have implications for mapping MA after stroke, with potential therapeutic applications.

Mapping selective neuronal loss and microglial activation in the salvaged neocortical penumbra in the rat.

Rescuing the ischemic penumbra from infarction is the mainstay of acute stroke therapy. However, the rescued penumbra may be affected by selective neuronal loss (SNL) and microglial activation (MA), which may hinder functional recovery and hence represent potential new therapeutic targets. Imaging them in vivo is currently attracting considerable interest, but relevant rat models are needed to underpin methods development and validation. Although striatal SNL/MA is well described following proximal MCA occlusion (MCAo), neocortical SNL/MA is still poorly characterized, yet has greater clinical relevance. This study aimed to assess the distribution and intensity of neocortical SNL and MA in a distal clip MCAo model known to cause severe neocortical ischemia. Spontaneously hypertensive rats were subjected to 45 min distal MCAo with ipsilateral common carotid artery occlusion. At day 14, post mortem SNL and MA were mapped using NeuN and OX42 immunohistochemistry, respectively. In a separate group, cerebral blood flow (CBF) was mapped during MCAo using (14)C-iodoantipyrine autoradiography. Values for SNL, MA, and CBF were obtained in the same set of anatomical ROIs covering the cortical MCA territory. Extensive SNL and MA affected the non-infarcted MCA cortex, adopting a well-defined regional distribution and a striking patchy/pseudo-columnar pattern. Regional intensities of SNL and MA were strongly inter-correlated, and also strikingly related to occlusion CBF, showing sharp rises for CBF <40%, i.e. the penumbra threshold. This rat model may be useful in providing in vitro reference for studies aiming to validate novel imaging tracers of SNL and MA in vivo.

T cell receptor complementarity determining region 3 length analysis reveals the absence of a characteristic public T cell repertoire in neonatal tolerance. The response in the "tolerant" mouse within the residual repertoire is quantitatively similar but qualitatively different.

All adult BALB/c mice immunized with hen egg white lysozyme (HEL) or its dominant determinant, peptide (p)106-116, mount a T cell response using a "public" Vbeta8.2Jbeta1.5 T cell clone. Neonatal exposure to tolerance-inducing doses of antigen can drastically diminish responsiveness in the draining lymph nodes but not in the spleens of animals challenged as adults with the cognate antigen. To determine the role of T cell deletion or anergy within the mechanisms of observed neonatal "tolerance," we treated neonatal BALB/c mice with HEL and directly followed the characteristic public clone using complementarity determining region 3 length T cell repertoire analysis. Our results confirm that despite intraperitoneal injection of neonates with a high dose of HEL emulsified in incomplete Freund's adjuvant, a strong splenic proliferative response to HEL was observed upon recall. However, the adult splenic T cell response of these neonatally treated mice lacked the usual Vbeta8.2Jbeta1.5 public clone characteristic of HEL-primed BALB/c mice. After challenge with HEL-complete Freund's adjuvant as adults, immunoglobulin (Ig)G2a isotype antibody was drastically reduced, and IgG1 was found to be the predominant anti-HEL IgG isotype expressed, indicating a deviation of cytokine response toward T helper type 2. 5-wk-old mice, nasally instilled with tolerogenic doses of HEL p106-116, also showed significant inhibition of this public T cell expansion. These results demonstrate that during neonatal and adult nasal tolerance induction, deletion/anergy removes the public clone, exposing a response of similar specificity but that is characterized by the T helper type 2 phenotype and a splenic residence.

A chronic 1 year assessment of MRI contrast agent-labelled neural stem cell transplants in stroke.

Non-invasive identification of transplanted neural stem cells in vivo by pre-labelling with contrast agents may play an important role in the translation of cell therapy to the clinic. Understanding the impact of these labels on the cells' ability to repair is therefore vital. In rats with middle cerebral artery occlusion (MCAo), a model of stroke, the transhemispheric migration of MHP36 cells labelled with the bimodal contrast agent GRID was detected on magnetic resonance images (MRI) up to 4 weeks following transplantation. However, compared to MHP36 cells labelled with the red fluorescent dye PKH26, GRID-labelled transplants did not significantly improve behaviour, and performance was akin to non-treated animals. Likewise, the evolution of anatomical damage as assessed by serial, T(2)-weighted MRI over 1 year indicated that GRID-labelled transplants resulted in a slight increase in lesion size compared to MCAo-only animals, whereas the same, PKH26-labelled cells significantly decreased lesion size by 35%. Although GRID labelling allows the in vivo identification of transplanted cells up to 1 month after transplantation, it is likely that some is gradually degraded inside cells. The translation of cellular imaging therefore does not only require the in vitro assessment of contrast agents on cellular functions, but also requires the chronic, in vivo assessment of the label on the stem cells' ability to repair in preclinical models of neurological disease.

Communication: Preparing undergraduate radiation therapy students for initial clinical patient interactions.

INTRODUCTION: Radiation therapy students need to demonstrate appropriate communication skills when entering the clinical environment. To assist students with preparation for their first clinical placement a clinical reasoning module comprising theory and practical sessions was developed. This paper describes the module and presents the results of student evaluations. METHODS: The module consisted of lectures, observational role-play and participatory role-play. Students were ultimately tasked with providing information to a simulated patient (SP). Each student received feedback independently from the SP, peers and facilitator. At the conclusion of the module, students had the opportunity to provide feedback via an anonymous survey (8 Likert scale questions with space for written comment). Data was analysed both quantitatively and qualitatively. RESULTS: Four hundred and thirty seven students were enrolled in the course between 2008 and 2016 and the response rate of the survey was 93%. Even though most students reported some level of anxiety before and during the role-play sessions, the majority of students perceived all aspects of the module to be extremely/very useful. The most useful aspect of the module (Likert scale assessment) was the feedback provided by the SP. The two most important themes arising from the thematic analysis were gaining an understanding of the role of the radiation therapist and the complexities of patient interactions. CONCLUSION: Overall, the module was deemed successful with students becoming conscious of newly acquired clinical knowledge whilst acknowledging patient feelings during interactions. Collaborative critiquing contributed to students' ability to self-reflect to improve clinical interactions.

The Development of Technology for Effective Respiratory-Gated Irradiation Using an Image-Guided Small Animal Irradiator.

The development of image-guided small animal irradiators represents a significant improvement over standard irradiators by enabling preclinical studies to mimic radiotherapy in humans. The ability to deliver tightly collimated targeted beams, in conjunction with gantry or animal couch rotation, has the potential to maximize tumor dose while sparing normal tissues. However, the current commercial platforms do not incorporate respiratory gating, which is required for accurate and precise targeting in organs subject to respiration related motions that may be up to the order of 5 mm in mice. Therefore, a new treatment head assembly for the Xstrahl Small Animal Radiation Research Platform (SARRP) has been designed. This includes a fast X-ray shutter subsystem, a motorized beam hardening filter assembly, an integrated transmission ionization chamber to monitor beam delivery, a kinematically positioned removable beam collimator and a targeting laser exiting the center of the beam collimator. The X-ray shutter not only minimizes timing errors but also allows beam gating during imaging and treatment, with irradiation only taking place during the breathing cycle when tissue movement is minimal. The breathing related movement is monitored by measuring, using a synchronous detector/lock-in amplifier that processes diffuse reflectance light from a modulated light source. After thresholding of the resulting signal, delays are added around the inhalation/exhalation phases, enabling the "no movement" period to be isolated and to open the X-ray shutter. Irradiation can either be performed for a predetermined time of X-ray exposure, or through integration of a current from the transmission monitor ionization chamber (corrected locally for air density variations). The ability to successfully deliver respiratory-gated X-ray irradiations has been demonstrated by comparing movies obtained using planar X-ray imaging with and without respiratory gating, in addition to comparing dose profiles observed from a collimated beam on EBT3 radiochromic film mounted on the animal's chest. Altogether, the development of respiratory-gated irradiation facilitates improved dose delivery during animal movement and constitutes an important new tool for preclinical radiation studies. This approach is particularly well suited for irradiation of orthotopic tumors or other targets within the chest and abdomen where breathing related movement is significant.

Phenytoin increases specific triacylglycerol fatty esters in skeletal muscle from horses with hyperkalemic periodic paralysis.

Previous studies have demonstrated that phenytoin decreases the levels of triacylglycerols in several tissues other than skeletal muscle. Since phenytoin is clinically effective in several skeletal muscle disorders, triacylglycerol metabolism in skeletal muscle from four normal Quarter horses and four Quarter horses with hyperkalemic periodic paralysis was examined. The horses with hyperkalemic periodic paralysis had low levels of 18:3 in the phospholipids, low levels of 16:0, 16:1 and 18:3 in the free fatty acids and low levels of 20:4 in triacylglycerols. Triacylglycerol levels were increased in skeletal muscle from seven (three controls, four hyperkalemic periodic paralysis) of the eight horses on treatment with oral phenytoin for one week. Instead of an increase in all fatty ester types only 16:0, 16:1, 18:1 and 18:2 were significantly increased. Total lipid phosphorus and the distribution of phospholipid fatty esters and free fatty acids were not significantly altered by phenytoin treatment in most cases. An alteration in triacylglycerol metabolism by phenytoin was also observed in primary cultures of normal equine skeletal muscle radiolabeled with 18:1, but not in those radiolabeled with 18:2. These findings suggest that phenytoin does not just increase the levels of triacylglycerol in skeletal muscle, but alters the utilization and incorporation of fatty esters. These findings suggest a potential involvement of triacylglycerol metabolism in the clinical efficacy of phenytoin in hyperkalemic periodic paralysis.

Investigation of single and paired measurements of adrenocorticotropic hormone for the diagnosis of pituitary pars intermedia dysfunction in horses.

BACKGROUND: Paired measurement of ACTH concentration may be more reliable than a single measurement. HYPOTHESIS/OBJECTIVES: To determine whether the mean of 2 measurements of ACTH concentration is more reliable in assessing pituitary pars intermedia dysfunction (PPID) than a single measurement. ANIMALS: Paired ACTH measurements were performed on (1) 148 occasions from 124 horses being investigated for PPID, (2) 90 occasions from 76 horses with PPID that were receiving treatment with pergolide, and (3) 63 occasions from 50 horses in which there was no clinical suspicion of PPID. Histologic examination of the pars intermedia was performed in 67 of the untreated horses. METHODS: Outcome of testing using single and the mean of paired samples was compared directly and both methods were compared against histology, which was considered the gold standard. RESULTS: Paired ACTH measurement altered binary classification as healthy or diseased in 6 of 211 cases, all off which had equivocal initial ACTH concentrations between 20 and 39 pg/mL. Using histology as the gold standard, optimal sensitivity and specificity for diagnosing PPID were 69.4 and 80.9%, respectively, for a single measurement and 72.2 and 76.2%, respectively, for paired measurements. The area under the receiver operating characteristic curve was 0.72 and 0.73 for single and paired measurements compared with histopathologic diagnosis, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Paired measurement of ACTH concentration offers no advantage over a single measurement.

Extension of nanoconfined DNA: Quantitative comparison between experiment and theory.

The extension of DNA confined to nanochannels has been studied intensively and in detail. However, quantitative comparisons between experiments and model calculations are difficult because most theoretical predictions involve undetermined prefactors, and because the model parameters (contour length, Kuhn length, effective width) are difficult to compute reliably, leading to substantial uncertainties. Here we use a recent asymptotically exact theory for the DNA extension in the "extended de Gennes regime" that allows us to compare experimental results with theory. For this purpose, we performed experiments measuring the mean DNA extension and its standard deviation while varying the channel geometry, dye intercalation ratio, and ionic strength of the buffer. The experimental results agree very well with theory at high ionic strengths, indicating that the model parameters are reliable. At low ionic strengths, the agreement is less good. We discuss possible reasons. In principle, our approach allows us to measure the Kuhn length and the effective width of a single DNA molecule and more generally of semiflexible polymers in solution.

Resolution of stroke deficits following contralateral grafts of conditionally immortal neuroepithelial stem cells.

BACKGROUND AND PURPOSE: Grafts of MHP36 cells have previously been shown to reduce dysfunction after global ischemia in rats. To test their efficacy after focal ischemia, MHP36 cells were grafted 2 to 3 weeks after transient intraluminal middle cerebral artery occlusion (tMCAO) in rats. METHODS: MHP36 cells were implanted into the hemisphere contralateral to the lesion, with 8 deposits of 3 microL of cell suspension (25 000 cells per microliter). Sham grafted rats received equivalent volumes of vehicle. Three groups, sham-operated controls (n=11), MCAO+sham grafts (n=10), and MCAO+MHP36 grafts (n=11), were compared in 3 behavioral tests. RESULTS: In the bilateral asymmetry test, MCAO+MHP36 grafted rats exhibited neglect before grafting but subsequently showed no significant dysfunction, whereas MCAO+sham grafted rats showed stable sensorimotor deficits over 18 weeks relative to controls. MCAO+sham grafted rats demonstrated spontaneous motor asymmetry and increased rotational bias after injection of dopamine agonists. MCAO+MHP36 and control groups exhibited no bias in either spontaneous or drug-induced rotation. In contrast to motor recovery, MCAO+MHP36 grafted rats showed no improvement relative to MCAO+sham grafted rats in spatial learning and memory in the water maze. MCAO produced large striatal and cortical cavitations in both occluded groups. Lesion volume was significantly reduced (P<0.05) in the MCAO+MHP36 grafted group. The majority of MHP36 cells were identified within the intact grafted hemisphere. However, MHP36 cells were also seen in the cortex, striatum, and corpus callosum of the lesioned hemisphere. CONCLUSIONS: MHP36 cells may improve functional outcome after MCAO by assisting spontaneous reorganization in both the damaged and intact hemispheres.

A temporal MRI assessment of neuropathology after transient middle cerebral artery occlusion in the rat: correlations with behavior.

The purpose of this study was to evaluate the temporal and spatial pathological alterations within ischemic tissue using serial magnetic resonance imaging (MRI) and to determine the extent and duration of functional impairment using objective behavioral tests after transient middle cerebral artery occlusion (tMCAO) in the rat. MRI signatures derived from specific anatomical regions of interest (ROI) were then appropriately correlated to the behavioral measures over the time course of the study (up to 28 days post-tMCAO). Sprague-Dawley rats (n = 12) were initially trained on the following behavioral tasks before surgery: bilateral sticky label test (for contralateral neglect); beam walking (for hindlimb coordination); staircase test (for skilled forelimb paw-reaching). Rats were then randomly assigned to receive either tMCAO (90 minutes, n = 6), by means of the intraluminal thread technique, or sham-control surgery (n = 6). Proton density, T2- and T2-diffusion-weighted MR images were acquired at 1, 7, 14, and 28 days post-tMCAO that were then smoothed into respective proton density, T2 relaxation, and apparent diffusion coefficient (ADC) maps. Apparent percent total lesion volume was assessed using T2W imaging. MR signatures were evaluated using the tissue maps by defining ROI for MCAO and sham-control groups, which corresponded to the caudate-putamen, forelimb, hindlimb, and lower parietal cortices both ipsilateral and contralateral to the occlusion site. Behavioral tests were undertaken daily from 1 to 28 days post-tMCAO. Results demonstrate that apparent percent lesion volume reduced from 1 to 7 days (P < 0.05) but then remained constant up to 28 days for the MCAO group. Pathological changes in the temporal profile of T2 and ADC tissue signatures were significantly altered in specific ROI across the time course of the study (P < 0.05 to <0.001), reflecting the progression of edema to necrosis and cavitation. Both T2 and ADC measures of ischemic pathology correlated with parameters defined by each of the functional tests (r > or =0.5, P < 0.05) across the time course. The staircase test revealed bilateral impairments for the MCAO group (P <0.001), which were best predicted by damage to the ipsilateral lower parietal cortex by means of hierarchical multiple regression analyses (R2 changes > or =0.21, P < or =0.03). Behavioral recovery was apparent on the beam walking test at 14 to 28 days post-MCAO, which was mirrored by MRI signatures within the hindlimb cortex returning to sham-control levels. This long-term study is the first of its kind in tracing the dynamic pathologic and functional consequences of tMCAO in the rat. Both serial MRI and objective behavioral assessment provide highly suitable outcome measures that can be effectively used to evaluate promising new antiischemic agents targeted for the clinic.

Neuroprotection in ischemia-reperfusion injury: an antiinflammatory approach using a novel broad-spectrum chemokine inhibitor.

Cerebral ischemia-reperfusion injury is associated with a developing inflammatory response with pathologic contributions from vascular leukocytes and endogenous microglia. Signaling chemokines orchestrate the communication between the different inflammatory cell types and the damaged tissue leading to cellular chemotaxis and lesion occupation. Several therapies aimed at preventing this inflammatory response have demonstrated neuroprotective efficacy in experimental models of stroke, but to date, few investigators have used the chemokines as potential therapeutic targets. In the current study, the authors investigate the neuroprotective action of NR58-3.14.3, a novel broad-spectrum inhibitor of chemokine function (both CXC and CC types), in a rat model of cerebral ischemia-reperfusion injury. Rats were subjected to 90 minutes of focal ischemia by the filament method followed by 72 hours of reperfusion. Both the lesion volume, measured by serial magnetic resonance imaging, and the neurologic function were assessed daily. Intravenous NR58-3.14.3 was administered, 2 mg/kg bolus followed by 0.5 mg/kg hour constant infusion for the entire 72-hour period. At 72 hours, the cerebral leukocytic infiltrate, tumor necrosis factor-alpha (TNF-alpha), and interleukin-8 (IL-8)-like cytokines were analyzed by quantitative immunofluorescence. NR58-3.14.3 significantly reduced the lesion volume by up to 50% at 24, 48, and 72 hours post-middle cerebral artery occlusion, which was associated with a marked functional improvement to 48 hours. In NR58-3.14.3-treated rats, the number of infiltrating granulocytes and macrophages within perilesional regions were reduced, but there were no detectable differences in inflammatory cell numbers within core ischemic areas. The authors reported increased expression of the cytokines, TNF-alpha, and IL-8-like cytokines within the ischemic lesion, but no differences between the NR58-3.14.3-treated rats and controls were reported. Although chemokines can have pro- or antiinflammatory action, these data suggest the overall effect of chemokine up-regulation and expression in ischemia-reperfusion injury is detrimental to outcome.

Cerebroprotective effect of the nitric oxide synthase inhibitors, 1-(2-trifluoromethylphenyl) imidazole and 7-nitro indazole, after transient focal cerebral ischemia in the rat.

The novel neuronal nitric oxide synthase inhibitors, 1-(2-trifluoromethylphenyl)imidazole (TRIM) and 7-nitro indazole (7-NI), were used to investigate the role of nitric oxide in a model of transient focal cerebral ischemia in vivo. In halothane-anesthetized rats, the middle cerebral artery (MCA) was occluded for 2 hours using an intravascular thread and then reperfused for 22 hours before histologic evaluation. TRIM (10, 20, or 50 mg/kg), 7-NI (60 mg/kg), TRIM (50 mg/kg) plus L-arginine (300 mg/kg), or L-arginine (300 mg/kg) alone was administered intraperitoneally, either at 5 or 90 minutes after MCA occlusion. Immediate administration (5 minutes after MCA occlusion) of TRIM produced a dose-related reduction in lesion size, which was reversed with L-arginine coadministration. Similarly, delayed administration of TRIM (90 minutes after MCA occlusion, 50 mg/kg) decreased total lesion volume by 48.4% +/- 13.0% in comparison to a reduction of 39.3% +/- 10.9% when TRIM (50 mg/kg) was administered immediately (5 minutes) after occlusion. 7-NI (60 mg/kg) reduced the total lesion volume by 38.5% +/- 13.7% when administered immediately (5 minutes) after MCA occlusion, but had no effect when administration was delayed (90 minutes). Neither TRIM (50 mg/kg) nor 7-NI (60 mg/kg), administered 5 minutes after MCA occlusion, had any significant effect on mean arterial blood pressure throughout the ischemic period or for up to 10 minutes after reperfusion. These results indicate that immediate or delayed administration of the selective neuronal NOS inhibitor TRIM reduces the lesion volume after transient MCA occlusion. In contrast, only immediate administration of 7-NI reduces lesion volume.

Incorporation of cells into an ELISA system enhances antigen-driven lymphokine detection.

The ability to measure successfully the levels of Th1 or Th2 cytokines during an in vitro antigen-driven, polyclonal T-cell response has proven to be more difficult than expected. Here we describe the development of a highly sensitive cell-based ELISA (celELISA) technique for the detection of murine Th1 and Th2 cytokines. The celELISA combines the quantification aspects of the conventional sandwich ELISA with the sensitivity of the ELISPOT assay. The celELISA was particularly useful for the improved detection of IL-2, IL-4, and to a lessor extent, IFN-gamma.

Residual public repertoires to self.

The consensus view about the constitution of the T cell receptor repertoire has shifted greatly even during this decade. Although the discovery of autoimmunity in the fifties had clearly shown that a repertoire must exist directed against self antigens, the extent of this repertoire was not fully appreciated. In our work we have tried to elucidate the nature of the antigenic specificities against which this self-directed repertoire is directed. The non-tolerized (residual) self-directed repertoire is a direct consequence of the hierarchy of antigenic determinant display, and is the most important influence in the organism's choice of which T cells to delete. Certain determinants remain "silent" and are neither displayed in the thymus nor in the periphery: these are a heterogeneous group which are invisible to T cells for a variety of reasons. One reason relates to the processing and presentation of determinants, and a second derives from the nature of the T cell receptor (TcR) and the avidity of the T cell for its target specificity.

Two distinct phases and mechanisms of axonal growth shown by primary vestibular fibres in the brain, demonstrated by parvalbumin immunohistochemistry.

Antibodies to parvalbumin label intensely a small number of non-overlapping fibre systems in embryonic rat brain. All are in hindbrain--the oculomotor and trochlear motor fibres, the acoustic and vestibular fibres of the VIIIth nerve, and an unidentified group of fibres which ascend under the dorsal surface in caudal medulla. Of these, the vestibular fibres are the first to acquire parvalbumin immunoreactivity, and we have used this property to follow the growth of their axons in the brain. This occurs in two phases. In the first, occurring at embryonic days 12-14, the axons grow in small groups or fascicles under the pial surface to their most distant terminal zones rostrally in the cerebellum and caudally in the descending vestibular nuclei. This growth is directed towards the two sites where germinal neuroepithelium is expanding over the medullary velum in forming the cerebellum and lateral recess of the IVth ventricle. In a second stage, commencing at E15, individual collaterals branch from these fascicles to arborize amongst their presumptive synaptic targets (cells of the vestibular nuclei and vestibulocerebellum) located in the sub-ventricular and ventricular layers. In this phase the axons follow a radial route, at right angles to their original subpial course, possibly by growing along radial glial processes. The target cells then migrate to their final position with the vestibular axons maintaining contact with them. The vestibular fibres are the first axons to enter the cerebellum, but from E15 onwards their fascicles are joined by increasing numbers of non-vestibular fibres following the same course. These other axons, and the movement of cells to form the deep cerebellar nuclei, separate the fascicles of vestibular fibres so that their course into the cerebellum becomes very diffuse. Thus this single set of axons grow, not only in two distinct phases, but also follow distinctly different substrates for growth in each. Furthermore, they then appear to act as pioneer fibres guiding the entry or egress of later-developing axons to or from the cerebellum.

Bicarbonate in the treatment of metabolic acidosis: effects on hepatic intracellular pH, gluconeogenesis, and lactate disposal in rats.

The effects of agents used in the treatment of metabolic acidosis could depend on the induced changes in intracellular pH (pHi). To determine the effect of sodium bicarbonate on hepatic pHi and function, this agent was infused into anesthetized rats with acute metabolic acidosis due to either diabetic ketoacidosis (DKA) or HCl infusion. Hepatic pHi was measured by 31P-magnetic resonance spectroscopy (MRS). A substantial increase in pHi occurred (from 7.13 +/- 0.08 to 7.32 +/- 0.08, P < .05) despite an increase in mixed venous PCO2. Isolated livers from normal rats or those with DKA were perfused at pH 6.8 and normal PCO2. With infusion of sodium bicarbonate, there was again an increase in pHi (delta pHi, + 0.27 +/- 0.06, P < .02) despite increases in both portal and hepatic venous PCO2. Lactate uptake was increased twofold to threefold (P < .001) by bicarbonate infusion in perfusions from both types of animals. Glucose output was increased twofold (P < .001) only in livers from normal animals.

The effects of sodium bicarbonate and a mixture of sodium bicarbonate and carbonate ("Carbicarb") on skeletal muscle pH and hemodynamic status in rats with hypovolemic shock.

Rats rendered hypotensive and acidotic by withdrawal of blood were treated by infusion of either an equimolar mixture of sodium bicarbonate and sodium carbonate ("Carbicarb"), sodium bicarbonate alone, or sodium chloride. Skeletal muscle intracellular pH (pHi) was measured using magnetic resonance spectroscopy from the chemical shift of the carbon-2 (C2) proton resonance of the imidazole ring of anserine. In the groups treated with alkali, arterial blood pH (pHa) was restored to normal, but no change was observed in the sodium chloride-treated animals. Despite an elevation of arterial blood partial pressure of CO2 (PaCO2) in the group treated with sodium bicarbonate, no significant change in pHi was observed in any group. Blood lactate levels, initially elevated in all groups, underwent only minor changes. In all three groups a transient and similar elevation of arterial blood pressure was observed after infusion. Differential effects of Carbicarb and sodium bicarbonate in metabolic acidosis may be dependent on the model of metabolic acidosis used, and an alteration in PaCO2 induced by alkali therapy may not be a major determinant of changes in pHi.

Breathing strategy of the adult horse (Equus caballus) at rest.

To investigate the mechanism underlying the polyphasic airflow pattern of the equine species, we recorded airflow, tidal volum, rib cage and abdominal motion, and the sequence of activation of the diaphragm, intercostal, and abdominal muscles during quiet breathing in nine adult horses standing at rest. In addition, esophageal, abdominal, and transdiaphragmatic pressures were simultaneously recorded using balloon-tipped catheters. Analysis of tidal flow-volume loops showed that, unlike humans, the horse at rest breathes around, rather than from, the relaxed volume of the respiratory system (Vrx). Analysis of the pattern of electromyographic activities and changes in generated pressures during the breathing cycle indicate that the first part of expiration is passive, as in humans, with deflation toward Vrx, but subsequent abdominal activity is responsible for a second phase of expiration: active deflation to below Vrx. From this end-expiratory volume, passive inflation occurs toward Vrx, followed by a second phase of inspiration: active inflation to above Vrx, brought about by inspiratory muscle contraction. Under these conditions the abdominal muscles appear to share the principal pumping duties with the diaphragm. Adoption of this breathing strategy by the horse may relate to its peculiar thoracoabdominal anatomic arrangement and to its very low passive chest wall compliance. We conclude that there is a passive and active phase to both inspiration and expiration due to the coordinated action of the respiratory pump muscles responsible for the resting adult horse's biphasic inspiratory and expiratory airflow pattern. This unique breathing pattern perhaps represents a strategy of minimizing the high elastic work of breathing in this species, at least at resting breathing frequencies.