Continuous evaluation of single-dose moxifloxacin concentrations in brain extracellular fluid, cerebrospinal fluid, and plasma: a novel porcine model.

BACKGROUND: Knowledge regarding CNS pharmacokinetics of moxifloxacin is limited, with unknown consequences for patients with meningitis caused by bacteria resistant to beta-lactams or caused by TB. OBJECTIVE: (i) To develop a novel porcine model for continuous investigation of moxifloxacin concentrations within brain extracellular fluid (ECF), CSF and plasma using microdialysis, and (ii) to compare these findings to the pharmacokinetic/pharmacodynamic (PK/PD) target against TB. METHODS: Six female pigs received an intravenous single dose of moxifloxacin (6 mg/kg) similar to the current oral treatment against TB. Subsequently, moxifloxacin concentrations were determined by microdialysis within five compartments: brain ECF (cortical and subcortical) and CSF (ventricular, cisternal and lumbar) for the following 8 hours. Data were compared to simultaneously obtained plasma samples. Chemical analysis was performed by high pressure liquid chromatography with mass spectrometry. The applied PK/PD target was defined as a maximum drug concentration (Cmax):MIC ratio >8. RESULTS: We present a novel porcine model for continuous in vivo CNS pharmacokinetics for moxifloxacin. Cmax and AUC0-8h within brain ECF were significantly lower compared to plasma and lumbar CSF, but insignificantly different compared to ventricular and cisternal CSF. Unbound Cmax:MIC ratio across all investigated compartments ranged from 1.9 to 4.3. CONCLUSION: A single dose of weight-adjusted moxifloxacin administered intravenously did not achieve adequate target site concentrations within the uninflamed porcine brain ECF and CSF to reach the applied TB CNS target.

Moyamoya disease in a European setting: a Danish population-based study.

BACKGROUND AND PURPOSE: The incidence of moyamoya disease (MMD) in Europe is not well known. In those affected, the risk of brain hemorrhage is considered low. The present study aimed to investigate the incidence and clinical presentation of MMD in the Danish population. METHODS: Eligible patients were identified in the Danish National Patient Register from 1994 to 2017. We collected clinical and radiological data from individual patient records from neurological, neurosurgical and paediatric units across Denmark. The diagnosis was validated according to established criteria. We also extracted basic demographic data on the cohort from the Danish Civil Registration System. RESULTS: A total of 52 patients fulfilled the diagnostic criteria for MMD. Most patients were native Danes and only 15% had an East Asian background. The ratio of female to male patients was 1.8, and the incidence had two peaks: one in childhood and another in young middle age. Until 2007, MMD was only diagnosed sporadically. From 2008 onwards, the incidence rate was 0.07 per 100 000 person-years (95% confidence interval 0.05-0.09 per 100 000 person-years). The most common mode of presentation was ischaemic stroke (33%), followed by hemorrhage (23%), headache (17%) and transient ischaemic attack (14%). CONCLUSIONS: Moyamoya disease is rare in Denmark, but is associated with a considerable risk of hemorrhage. Thus, MMD should be considered in the evaluation for ischaemic as well as hemorrhagic stroke paediatric and middle-aged Caucasians.

Wistar rats subjected to chronic restraint stress display increased hippocampal spine density paralleled by increased expression levels of synaptic scaffolding proteins.

The aim of this study was to investigate whether the previously reported effect of chronic restraint stress (CRS) on hippocampal neuron morphology and spine density is paralleled by a similar change in the expression levels of synaptic scaffolding proteins. Adult male Wistar rats were subjected either to CRS (6 h/day) for 21 days or to control conditions. The resulting brains were divided and one hemisphere was impregnated with Golgi-Cox before coronal sectioning and autometallographic development. Neurons from CA1, CA3b, CA3c, and dentate gyrus (DG) area were reconstructed and subjected to Sholl analysis and spine density estimation. The contralateral hippocampus was used for quantitative real-time polymerase chain reaction and protein analysis of genes associated with spine density and morphology (the synaptic scaffolding proteins: Spinophilin, Homer1-3, and Shank1-3). In the CA3c area, CRS decreased the number of apical dendrites and their total length, whereas CA1 and DG spine density were significantly increased. Analysis of the contralateral hippocampal homogenate displayed an increased gene expression of Spinophilin, Homer1, Shank1, and Shank2 and increased protein expression of Spinophilin and Homer1 in the CRS animals. In conclusion, CRS influences hippocampal neuroplasticity by modulation of dendrite branching pattern and spine density paralleled by increased expression levels of synaptic scaffolding proteins.

Hypothalamic deep brain stimulation influences autonomic and limbic circuitry involved in the regulation of aggression and cardiocerebrovascular control in the Göttingen minipig.

BACKGROUND: Deep brain stimulation (DBS) in the ventral tuberal hypothalamus (VTH) is currently under investigation for the treatment of severe obesity. Stimulation impact on a number of closely related hypothalamic neural systems could potentially influence normal hypothalamic function and thereby generate adverse side effects. OBJECTIVE: To assess the feasibility and safety of VTH DBS in a non-primate large animal model. METHODS: In the VTH of 6 Göttingen minipigs, quadropolar leads were implanted bilaterally (n = 2) or unilaterally (n = 4), using optimized MRI sequences allowing identification of major diencephalic landmarks. Heart rate, weight, behavior and nighttime locomotor activity were recorded throughout the study period. Two of the unilaterally implanted minipigs were examined with [15O]H2O positron emission tomography (PET) scans performed in DBS-off and DBS-on mode. RESULTS: VTH DBS elicited an amplitude-dependent increase in heart rate and transient aggressive behavior. PET demonstrated that VTH DBS caused a global increase in cerebral blood flow velocities and decreased mean transit time. CONCLUSIONS: VTH DBS results in behavioral and physiological changes, which may derive from activation of closely related limbic and autonomic networks. Caution and further studies of longer length should be requested before this procedure is used more widely in humans.

Regional brain atrophy in primary fatigued patients with multiple sclerosis.

OBJECTIVE: To examine the relationship between fatigue, regional brain atrophy and normal appearing white matter damage in patients with multiple sclerosis. METHODS: Primary fatigued (PF) (n, 17) and non-fatigued (NF) (n, 17) patients with relapsing remitting multiple sclerosis and moderate disability were grouped according to their subjective fatigue score. Also, they were examined with respect to processing speed and central motor activation during isometric contraction. Using 3 Tesla MRI quantitative analyses were performed on normal appearing brain tissue and of brain structure volumes with tensor based morphometry. RESULTS: Between the PF and NF patients there was no significant differences in brain parenchymal fraction (81.5% vs. 82.4%), lesion load (0.53% vs. 0.36%) and NAA/Cr ratio (1.29 vs. 1.32 respectively). Eleven clusters of atrophy in PF versus NF involved gray and nearby white matter, the majority being located in areas functionally related to attentional control. Central motor activation was associated with atrophy in five regions in PF patients, three clusters involving the premotor and primary motor cortex. Normal appearing white matter did not differ between groups. CONCLUSION: Primary fatigued patients with multiple sclerosis have extended regional atrophy of supratentorial brain parenchyma, involving the cerebral cortex, nearby white matter and the caudate head, areas which are functionally related to attentional control. We suggest that impaired central motor activation is due to interruption of the cortico-subcortical motor circuits involving the motor cortex.

Neuromodulation in a minipig MPTP model of Parkinson disease.

Large animal neuroscience enables the use of conventional clinical brain imagers and the direct use and testing of surgical procedures and equipment from the human clinic. The greater complexity of the large animal brain additionally enables a more direct translation to human brain function in health and disease. Economical, ethical, scientific and practical issues may on the other hand hamper large animal neuroscience. Large animal neuroscience should therefore either be performed in order to examine large animal species dependent problems or to complement promising small animal basic studies by constituting an intermediate research system, bridging small animal CNS research to the human CNS. We have, accordingly, during the last ten years used the Gottingen minipig to examine neuromodulatory treatment modalities such as stem cell transplantation and deep brain stimulation directed towards Parkinson disease. This has been accomplished by the development of a MPTP-based large animal model of Parkinson disease in the Gottingen minipig and the development of stereotaxic and surgical approaches needed to manipulate the Gottingen minipig CNS. The instituted changes in the CNS can be evaluated in the live animal by brain imaging (PET and MR), cystometry, gait analysis, neurological evaluation and by post mortem examination based on histology and stereological analysis.

Chronic subthalamic high-frequency deep brain stimulation in Parkinson's disease--a histopathological study.

This study describes the pathological findings in the brain of a patient with Parkinson's disease (PD) treated with bilateral subthalamic high-frequency deep brain stimulation (STN DBS) for 29 months prior to death. After routine neuropathological examination, tissue blocks containing the electrode tracts, the subthalamic nucleus (STN), the substantia nigra and the pre-frontal cortex were paraffin embedded and cut into 5-microm-thick serial sections and stained with several conventional staining methods and immunohistochemistry. Bilateral nigral depigmentation, cell loss and Lewy body formation confirmed the diagnosis of PD. Microscopic evaluation furthermore confirmed the location of the electrodes in the STN. The electrode tracts were surrounded by a 150-microm-wide glial fibrillary acidic protein (GFAP)-positive capsule consisting of a thin collagen layer lining the lumen of the tract, whilst an area with few cells and axons constituted the capsule wall towards the surrounding normal brain tissue. The brain tissue appeared normal outside the capsule boundaries with no difference in areas of stimulation compared with areas of no stimulation. Our results correspond with previous studies performed after fewer months of STN DBS and indicate mild histopathological changes in the vicinity of the electrode tract, appearing to result from the electrode placement and not from the electrical stimulation.

Does chronic low-dose treatment with cyclosporine influence the brain? A histopathological study in pigs.

Twenty percent of the patients immunosuppressed with cyclosporine A (CsA) develop neurological side effects such as tremor, paresthesias, headache, seizures, visual disorders, paresis, and coma-CsA encephalopathy. The encephalopathy resolves on CsA discharge; autopsies of recovered patients are normal. Characteristic lesions are seen on magnetic resonance imaging (MRI) during the period of encephalopathy. MRI of asymptomatic patients receiving CsA as well as most recovered patients are normal. Several theories of pathogenesis have been proposed, but none has been firmly established. The current placebo-controlled study, blinded to the investigator, was accordingly initiated to elucidate histopathological changes in the brain. Twelve adult Göttingen minipigs were randomized into two groups treated with either low-dose CsA (10 mg/kg/d) or no treatment for 6 months. Behavior, blood pressure, and blood parameters were measured throughout the study. All animals had a cerebral MRI before sacrifice. Three control pigs and one CsA-treated pig died during observation and were excluded from the study. None of the remaining eight pigs displayed behavioral signs or MRI-visible lesions characteristic of CsA encephalopathy. The brains appeared all normal on the gross pathological examination, but microscopy revealed perivascular, meningeal, and neuronal tissue infiltration with granulocytes and mononuclear cells in one CsA-treated pig, while the remaining pigs were without histopathological lesions. Pathological changes were noticed in one out of five CsA-treated animals, corresponding to the percentage of patients treated with CsA who develop CsA encephalopathy. To pursue this finding, two studies, one using CsA 20 mg/kg/d for 6 months and one using CsA 10 mg/kg/d for 12 months, have been initiated.

Distribution and morphology of serotonin-immunoreactive neurons in the brainstem of the New Zealand white rabbit.

The aim of the present study was to demonstrate the morphology and distribution of the serotonergic neurons in the brainstem of the New Zealand white rabbit by using a highly specific immunocytochemical procedure. It was possible to divide the serotonergic neurons into a rostral group, which is situated in the mesencephalon and the rostral part of the pons containing four serotonergic nuclei, and a caudal group, which is located in the medulla and the caudal part of the pons containing five serotonergic nuclei. The localization of the serotonergic neurons is presented in a detailed brainstem atlas, and the distribution of the serotonergic neurons is in accordance with results obtained by other authors in different species. Special emphasis was given to the fact that many of the serotonergic neurons were distributed in more lateral parts of the brainstem. The laterally orientated neurons, which were large and multipolar, were morphologically different from the serotonergic neurons in the midline, which were mostly small and relatively nonpolar. The serotonergic system of the New Zealand white rabbit has undergone a major lateralization, like the serotonergic system of man and higher primates, and it may therefore be excellently suited for experimental procedures directed towards the serotonergic system. The difference between serotonergic neurons localized in the midline and those situated laterally may reflect functional differences based on dissimilarity in connectivity and morphology, and this possible subspecialization of the serotonergic system is discussed in the context of present knowledge of serotonergic anatomy and function.

An autometallographic technique for myelin staining in formaldehyde-fixed tissue.

A new autometallographic (AMG) technique for staining myelin in formaldehyde- or paraformaldehyde- (PFA) fixed tissue is presented. The tissue sections were exposed to AMG development without prior treatment with silver salts. The method was examined on PFA-fixed tissue from mouse, rat, pig, and formaldehyde-fixed human autopsy material. Samples from brain, spinal cord, cranial, and spinal nerves were either cut on a vibratome, frozen and cryostat sectioned, or embedded and microtome sectioned, before AMG development and counterstaining. The AMG-myelin technique results in a specific black/dark-brown staining of myelin in all parts of the CNS and PNS. It works on all species examined, independent of the histological preparation techniques applied. The AMG staining is stable, stays unchanged through decades, allows counterstaining, and has previously been used with immunohistochemical techniques. On perfusion-fixed tissue the technique works without further fixation, but the intensity of the AMG-myelin staining is increased by increased postfixation time. Additionally, immersion fixation has to last for days depending on the size of the tissue block in order to obtain proper myelin staining. The most feasible explanation of the chemical events underlying the AMG-myelin technique is that nano-sized clusters of metallic silver are formed in the myelin as a result of chemical bounds with reducing capacity, exposed or created by the formaldehyde molecule. The AMG method is simple to perform and as specific as the conventional osmium and luxol fast blue stainings. The present technique is thus an effective, simple, inexpensive, and quick myelin staining method of formaldehyde- or PFA-fixed tissue.

New strategies for embedding, orientation and sectioning of small brain specimens enable direct correlation to MR-images, brain atlases, or use of unbiased stereology.

We present a newly developed brain slicing machine and technique for tissue embedding, which enable orientation of fresh or fixed brain tissue from small laboratory animals, in any given position, and subsequent tissue sectioning into slabs with an optional thickness between 0.5 and 20 mm. The oriented tissue slabs may be analysed directly, or processed further on a cryostat or vibratome, into thin stainable histological sections, and aligned to MR-images or brain atlases, depending on the reference used for the initial orientation. Additionally, we describe a new embedding medium (HistOmer) which is an alginate cold polymer ready for instant use after mixing with water. HistOmer allows accurate positioning of the tissue during embedding, and at the same time supports and protects the embedded tissue during sectioning. HistOmer is, therefore, described comprehensively and compared with other commonly used embedding media. This novel slicing technique may also, as illustrated, be used to perform isotropic random orientation of the embedded tissue, before sectioning into tissue slabs of the same thickness. The technique thereby fulfills the requirements for optimal tissue sampling and preparation needed for an unbiased stereologic analysis.

Determination of stereotaxic coordinates for the hippocampus in the domestic pig.

In this study a stereotaxic instrument and a stereotaxic procedure based on external skull structures, to be used in prepubertal male Landrace pigs weighing less than 30 kg, is described. The instrument represents an adaptation of the apparatus designed by Marcilloux et al., Brain Res Bull 1989;22:591-597, but we have modified the instrument for stereotaxic procedures based on external skull structures, instead of intracerebral structures necessitating ventriculography (Marcilloux et al., Brain Res Bull 1989;22:591-597). For this reason the U-shaped frame and the ear-bar supports have been changed allowing the three-dimensional placement of the ear-bars into the oblique auditory canals. Firm fixation of the skulls of pigs weighing less than 30 kg, was furthermore secured with modified infraorbital ridges and hard palate pieces. Measurements of distances between external skull structures in animals of the same sex, age and weight showed a negligible variation, thus enabling definition of the horizontal, frontal and sagittal zero planes using external skull structures alone. Stereotaxic coordinates for the hippocampal region of male Landrace pigs weighing 10 kg were then provided and the coordinates from two different levels of the hippocampal region are presented in the text. The reliability of the stereotaxic instrument was finally secured by intrahippocampal injections of ink at predetermined coordinates.

Oriented sectioning of irregular tissue blocks in relation to computerized scanning modalities: results from the domestic pig brain.

We present a new method allowing direct comparison between images obtained by present digital scanning modalities and histological sections from the same object. More specifically the paper illustrates how to orientate, embed, and section large irregular tissue blocks after magnetic resonance imaging (MRI) in such a way that accurate correlation of the digital data sets to histological sections is possible. The functionality and capability of the described procedure and slicing machine is illustrated by results from the pig brain. Accordingly, three pigs were MR-scanned, followed by perfusion fixation. The brains were removed, oriented according to the MR scans, embedded in alginate, and cut on a newly developed slicing machine. The tissue blocks were then stained to reveal grey and white matter and photographed before final sectioning on a cryostat into 80 microm thick sections which were Nissl-stained with toluidine. The results demonstrate how our method enables direct comparison between the pig brain MR images and the later obtained histological sections. The alginate embedding method and slicing machine offer the same possibilities for other parenchymateous organs and soft tissues and may, in addition, be of use in stereological analysis.

A MRI-compatible stereotaxic localizer box enables high-precision stereotaxic procedures in pigs.

We present a nonmagnetic Plexiglas stereotaxic localizer box that can be fitted directly to the pig skull by aluminum screws, allowing stereotaxic MRI or ventriculography and subsequent high-precision stereotaxic procedures. The localizer box was used to target the subthalamic nucleus (STN) bilaterally in five female Göttingen minipigs. Stereotaxic markers were inserted in the pig skull, the head fixated in the localizer box by aluminum screws inserted bilaterally in the zygoma bone with the hard palate locked on a horizontal palate holder. MRI was obtained on a 3T-MR-imager revealing the relation between the inserted markers and the estimated STN-position, and thus the target coordinates. After the MRI, a stereotaxic frame with attached micromanipulator was locked on to the localizer box converting it into a stereotaxic device. The stereotaxic markers were exposed and used as starting point for the stereotaxic procedure, whereby a microelectrode for electrolytic lesioning was inserted in the STN. Postmortem histological analysis revealed 70% correct STN-targeting. The average distance from the lesion center to the STN center was 1.2 mm with a S.D. of 1.1 mm. The most displaced lesion being 3.6 mm from the STN center. We conclude that the described localizer box secure firm head fixation, allowing stereotaxic MRI and subsequent conversion into a stereotaxic device for high-precision stereotaxic procedures.

Development of neuromodulation treatments in a large animal model--do neurosurgeons dream of electric pigs?

The Göttingen minipig has been established as a translational research animal for neurological and neurosurgical disorders. This animal has a large gyrencephalic brain suited for examination at sufficient resolution with conventional clinical scanning modalities. The large brain, further, allows use of standard neurosurgical techniques and can accommodate clinical neuromodulatory devises such as deep brain stimulation (DBS) electrodes and encapsulated cell biodelivery devices making the animal ideal for basic scientific studies on neuromodulation mechanisms and preclinical tests of new neuromodulation technology for human use. The use of the Göttingen minipig is economical and does not have the concerns of the public associated with the experimental use of primates, cats, and dogs, thus providing a cost-effective research model for translation of rodent data before clinical trials are initiated.

Evidence implicating BRD1 with brain development and susceptibility to both schizophrenia and bipolar affective disorder.

Linkage studies suggest that chromosome 22q12-13 may contain one or more shared susceptibility genes for schizophrenia (SZ) and bipolar affective disorder (BPD). In a Faeroese sample, we previously reported association between microsatellite markers located at 22q13.31-qtel and both disorders. The present study reports an association analysis across five genes (including 14 single nucleotide and two microsatellite polymorphisms) in this interval using a case-control sample of 162 BPD, 103 SZ patients and 200 controls. The bromodomain-containing 1 gene (BRD1), which encodes a putative regulator of transcription showed association with both disorders with minimal P-values of 0.0046 and 0.00001 for single marker and overall haplotype analysis, respectively. A specific BRD1 2-marker 'risk' haplotype showed a frequency of approximately 10% in the combined case group versus approximately 1% in controls (P-value 2.8 x 10(-7)). Expression analysis of BRD1 mRNA revealed widespread expression in mammalian brain tissue, which was substantiated by immunohistochemical detection of BRD1 protein in the nucleus, perikaryal cytosol and proximal dendrites of the neurons in the adult rat, rabbit and human CNS. Quantitative mRNA analysis in developing fetal pig brain revealed spatiotemporal differences with high expression at early embryonic stages, with intense nuclear and cytosolar immunohistochemical staining of the neuroepithelial layer and early neuroblasts, whilst more mature neurons at later embryonic stages had less nuclear staining. The results implicate BRD1 with SZ and BPD susceptibility and provide evidence that suggests a role for BRD1 in neurodevelopment.

New strategies for the treatment of Parkinson's disease hold considerable promise for the future management of neurodegenerative disorders.

Neurodegenerative diseases are often considered incurable with no efficient therapies to modify or halt the progress of disease, and ultimately lead to reduced quality of life and to death. Our knowledge of the nervous system in health and disease has, however, increased considerably during the last fifty years and today, neuroscience reveals promising new strategies to deal with disorders of the nervous system. Some of these results have been implemented with success in the treatment of Parkinson's disease, a common neurodegenerative illness affecting approximately 1% of the population aged seventy or more. Parkinson's disease is characterized by a massive loss of dopaminergic neurons in the substantia nigra, leading to severe functional disturbance of the neuronal circuitry in the basal ganglia. A thorough description of basal ganglia circuitry in health and disease is presented. We describe how the functional disturbances seen in Parkinson's disease may be corrected at specific sites in this circuitry by medical treatment or, in advanced stages of Parkinson's disease, by neurosurgical methods. The latter include lesional surgery, neural transplantation and deep brain stimulation, together with future treatment strategies using direct or indirect implantation of genetically modified cell-lines capable of secreting neurotrophic factors or neurotransmitters. Advantages and disadvantages are briefly mentioned for each strategy and the implications for the future and the possible use of these interventions in other neurodegenerative diseases are discussed, with special emphasis on deep brain stimulation.

Effects of high frequency deep brain stimulation on urine storage and voiding function in conscious minipigs.

AIMS: Suprapontine neural integration during the storage phase is decisive for the timing of voiding. Neurological disorders like Parkinson's disease are thus frequently complicated by bladder dysfunction. The aim of the present study was to investigate the effect of high frequency deep brain stimulation on the urine storage and voiding function in conscious Parkinsonian minipigs. MATERIALS AND METHODS: Five Goettingen minipigs had a Parkinsonism-like state induced by intoxication with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). An electrode for chronic stimulation was placed unilaterally in the subthalamic nucleus. The effect of stimulation was determined by the outcome of transurethral cystometries performed with the stimulation in on- and off-mode. RESULTS: Of 20 planned cystometry-sessions 18 were completed. Six incomplete voidings occurred in stimulation on-mode and five in off-mode. Interruption of the stimulation for 2 days was followed by a significant increase in pressure rise on filling to cystometric capacity, from 7 to 21 cmH(2)O (P = 0.005), and an insignificant reduction in cystometric capacity from 30 to 26 ml/kg bodywt. (P = 0.370), leading to a significant decrease in bladder compliance from 124 to 34 ml/cmH(2)O (P = 0.013). CONCLUSIONS: Transurethral cystometry was a feasible examination technique in pigs. The findings demonstrate that high frequency deep brain stimulation changes the bladder characteristics in the storage phase. Since bladder pressure and capacity responded differently to interruption of stimulation distinct neural mechanisms must be involved in the modulation of sensory information on bladder tension and stretch.