A population study on the epidemiology and outcome of brain injuries in intensive care.

BACKGROUND: Traumatic Brain Injury (TBI) is characterized by a highly heterogenous profile in terms of pathophysiology, clinical presentation and outcome. OBJECTIVE: This is the first population study investigating the epidemiology and outcomes of moderate-to-severe TBI in Cyprus. Patients treated in the Intensive Care Unit (ICU) of Nicosia General Hospital, the only Level 1 Trauma Centre in the country, were recruited between January 2013 and December 2016. METHODS: This was an observational cohort study, using longitudinal methods and six-month follow-up. Patients (N = 203) diagnosed with TBI were classified by the Glasgow Coma Scale at the Emergency Department as moderate or severe. RESULTS: Compared to international multicentre studies, the current cohort demonstrates a different case mix that includes older age, more motor vehicle collisions and lower mortality rates. There was a significantly higher proportion of injured males. Females were significantly older than males. There were no sex differences in the type, severity or place of injury. Sex did not yield differences in mortality or outcomes or on injury indices predicting outcomes. In contrast, older age was a predictor of higher mortality rates and worse outcomes. CONCLUSION: Trends as described in the study emphasize the importance of continuous evaluation of TBI epidemiology and outcome in different countries.

Psychological support for families of ICU patients: longitudinal documentation of the service.

This study is the first systematic effort to investigate psychological services provided to relatives of ICU patients at Nicosia General Hospital. Documentation of psychological sessions provided to relatives of ICU patients for the years 2011-2014 was analyzed. To investigate possible differences in the total number of sessions for the referenced years, the records were analyzed using patients' demographics, the outcome of hospitalization and the total number of sessions with relatives. A questionnaire was sent to the ICU staff aiming to identify their perception towards the need for psychological support. A total number of 863 psychological sessions were conducted with 640 relatives of 345 patients hospitalized in the ICU. Results indicate that more sessions are recorded when the outcome of younger patients' condition worsens, whereas the number of sessions decreases for older patients' families. When comparing the personnel's beliefs, regarding the importance of providing psychological services to different age groups, significant difference was found suggesting that the older the patient the less sessions they believe are required indicating a possible ageism bias.

Late-onset neurodegeneration in mice with increased dosage of the proteolipid protein gene.

Mutations of the proteolipid protein (Plp) gene cause a generalized central nervous system (CNS) myelin deficit in Pelizaeus-Merzbacher disease of man and various tremor syndromes in animal models. X-linked spastic paraplegia is also due to Plp gene mutations but has a different clinical profile and more restricted pathology involving specific tracts and regions. We have shown previously that PLP overexpression in mice homozygous for a Plp transgene results in premature arrest of CNS myelination and premature death. Here, we demonstrate that a low-level increase in Plp gene expression in transgenic mice causes significant axonal degeneration and demyelination with predilection for specific tracts. Following normal motor development, aged mice develop progressive myelin loss, axonal swellings with resultant Wallerian degeneration, and marked vacuolation of the neuropil associated with ataxia, tremor, and seizures. The age of onset and severity of the phenotype is a function of Plp gene dosage. The corticospinal tracts, optic nerve, fasciculus gracilis cerebellum, and brainstem are particularly involved. Although oligodendrocyte cell bodies show little abnormality, their inner adaxonal tongue is often abnormal, suggesting a perturbation of the axon/glial interface that may underlie the axonal changes. We conclude that abnormal expression of an oligodendrocyte-specific gene can cause axonal damage, a finding that is relevant to the pathogenesis of PLP-associated disorders and probably to other myelin-related diseases.

Hindshaker, a novel myelin mutant showing hypomyelination preferentially affecting the spinal cord.

Animals with spontaneous mutations affecting myelin formation have provided useful information about the genetic and cellular mechanisms regulating normal and abnormal myelination. In this paper we describe a novel murine mutation termed hindshaker (hsh), which is inherited in an autosomal recessive manner. Affected mice are characterised by a variable tremor of the hind end which commences at about 2 weeks of age and largely disappears in animals older than 6 weeks. There is hypomyelination affecting predominantly the spinal cord, although the optic nerves and brain are involved to a much lesser degree. The defect of thinly myelinated and naked axons is maximal at 20 days of age and largely resolves with time so that in the adult most axons are myelinated. The myelin structure appears normal and immunostains for the major proteins. Although the distribution of oligodendrocytes in the spinal cord is similar to normal during the period of hypomyelination, there are fewer mature cells. The hsh mutation appears to delay the maturation of oligodendrocytes, particularly in the spinal cord. Additionally, there is a considerable variation in phenotypic expression and in penetrance when the mutation is expressed on different genetic backgrounds, suggesting the hsh locus is subject to the influence of modifying gene(s). Identification of the hsh gene should identify a factor important in the development of oligodendrocytes, particularly those in the spinal cord.

Expression of the dm-20 isoform of the plp gene in olfactory nerve ensheathing cells: evidence from developmental studies.

The olfactory bulb is a specialized area of the CNS with well-defined areas containing both myelinated, and non-myelinated axons. The presence of plp gene transcripts has been demonstrated previously in the olfactory bulb, but no detailed description of plp gene activity in this complex area of the CNS is available. In this study we describe the developmental expression of the two plp gene isoforms, plp and dm-20, and their products in the mouse olfactory bulb. plp gene activity was present in the non-myelinated olfactory nerve layer of the bulb from E14 through to adult ages. Expression in the deeper layers of the bulb, associated with myelination of the second order axons, was apparent from P10 onwards. dm-20 was the predominant isoform expressed at the transcript level in the olfactory nerve layer and was the only isoprotein demonstrable by immunostaining. This expression was associated with the resident glial cell of the non-myelinated olfactory nerve layer, the olfactory nerve ensheathing cell. Selective expression of the DM-20 isoprotein in the non-myelin forming olfactory nerve ensheathing cells implies a role for DM-20 other than as a structural myelin protein. Further study of this specialized glial cell may prove useful in elucidating the specific functions(s) of the DM-20 isoprotein.

Evidence that some oligodendrocyte progenitors in the developing optic pathway express the plp gene.

DM-20, a product of the proteolipid protein (plp) gene, has been demonstrated in the spinal cord of the mouse embryo as early as embryonic day 12 (E12) in certain cells, some of which are identifiable as oligodendrocyte progenitors. The present work uses optic pathways of rat and mouse as well-characterized systems for the study of gliogenesis. plp gene expression was monitored with a combination of reverse transcriptase polymerase chain reaction, in situ hybridization, and immunostaining with antibodies to different PLP peptide sequences, combined with O-2A lineage markers. In tissue sections, hybridizing cells were detected initially in the proximal optic tracts between E18 and birth and thereafter progressively in the chiasm and optic nerves. Small unbranched cells expressing DM-20 but not myelin basic protein (MBP) and probably representing progenitors were detectable by immunostaining in similar locations. With increasing postnatal ages, cells representing maturing oligodendrocytes which co-label for PLP and MBP are present in the optic pathways. In vitro analysis of freshly dissociated cells from premyelinated optic nerve demonstrated that the plp gene is expressed in some O-2A progenitor cells as well as mature oligodendrocytes. We also present evidence that increase in expression of the plp gene along the O-2A lineage differentiation is not progressive but that downregulation at the proligodendroblast (O4+/O1-) stage probably occurs. We suggest that progenitors express the dm-20 isoform while oligodendrocytes express predominantly the plp isoform. Not all progenitors express the plp gene at the times studied, indicating that the presence of DM-20 is either transitory in individual cells or that only a sub-population is involved. The function of DM-20 at this early stage of the oligodendrocyte lineage has yet to be determined.

Oligodendrocyte progenitors in the embryonic spinal cord express DM-20.

Oligodendrocyte progenitors, originating in the ventral ventricular zone of the embryonic rodent spinal cord, migrate and differentiate into the oligodendrocytes myelinating the future white matter. Transcripts for the dm-20 isoform of the proteolipid protein (plp) gene are detectable initially in cells of the ventral ventricular region of the embryonic central canal and subsequently throughout the white matter. The dm-20+ cells are present several days before oligodendrocytes or myelin sheaths are detectable. The purpose of the present study was to determine if DM-20 protein is present and whether DM-20+ cells can be linked to the oligodendrocyte lineage in the mouse spinal cord. Expression of plp and dm-20 transcripts and product was monitored using reverse transcription polymerase chain reaction (RT-PCR), and in situ hybridization and immunostaining of cryosections and associated cultures. Cell identification was performed using antigenic markers characterizing different stages of oligodendrocyte differentiation. We show a temporal and spatial progression of cells expressing dm-20 transcripts and product from the ventral ventricular zone at embryonic day 13 (E13.0), via the lateral borders of the floor plate to the ventral pia and white matter. The cells, initially devoid of myelin basic protein (MBP) and PLP, co-express these myelin proteins at approximately E16.5/17.0. Some DM-20+ cells co-label with definitive markers of the early oligodendrocyte lineage, are capable of mitosis and subsequently differentiate into oligodendrocytes. Other DM-20+ cells may represent earlier precursor cells. The expression of DM-20 in oligodendrocyte progenitors is consistent with a postulated role in glial cell development.

Immunocytochemical and lectin histochemical study of neuronal lesions in autonomic ganglia of horses with grass sickness.

Equine grass sickness (EGS) is a primary dysautonomia characterised pathologically by lesions in autonomic ganglia, enteric plexi and specific nuclei in the CNS. Immunocytochemistry and lectin histochemistry of the autonomic ganglia were used to determine whether abnormalities can be detected in specific proteins or cellular organelles. EGS ganglia contained a mixture of morphologically normal and abnormal neurons, the former appearing identical to cells from control animals. Affected cells showed marked disturbances in neurofilament (NF) proteins and beta-tubulin, major components of the cytoskeleton; in most neurons immunoreactivity was reduced or absent while the distribution was altered in the remainder. Staining for neuron-specific enolase, a pan-neuronal marker, was severely reduced or absent, as was reactivity for the catecholaminergic enzyme tyrosine hydroxylase. However, affected neurons showed a marked increase in dopamine-beta-hydroxylase (D beta H), another enzyme associated with noradrenaline synthesis. Wheat germ agglutinin and Griffonia simplicifolia B4 lectin histochemistry was used to label membranes of the Golgi apparatus, which stained as discrete curvilinear perinuclear profiles. All affected neurons showed abnormalities with either complete loss of reaction or amorphous centrally located lectin staining. The results indicate perturbation in a wide variety of cytoplasmic and cytoskeletal proteins. In the majority of instances there is a decrease in stainable protein; the increase in D beta H may indicate a failure to be transported down the axon with resultant accumulation in the perikaryon. Loss of a recognisable Golgi structure appears to be an early event in the neuropathology of EGS.

In vitro studies of axonally-regulated Schwann cell genes during Wallerian degeneration.

Wallerian degeneration in vivo is associated with marked downregulation of myelin protein genes such as P(o) and upregulation of other genes such as nerve growth factor receptor (NGF-R), glial fibrillary acidic protein (GFAP) and neural cell adhesion molecule (N-CAM). This study examines the expression of these genes during Wallerian degeneration in vitro and how manipulating Ca2+ affects this response. Small explants of sciatic nerve from normal young adult rats cultured for five days show similar reversal of the myelinating phenotype as found in vivo. If Ca++ is removed from the culture medium through the addition of EGTA, expression of the nerve growth factor receptor and glial fibrillary acidic protein genes is inhibited but downregulation of the P(o) gene still occurs. Explants cultured in medium containing EGTA are still capable of expressing nerve growth factor receptor if the medium is replaced by one containing Ca2+. Supplementation of normal medium with drugs modulating Ca2+, such as Bepridil which blocks the Na+Ca2+ exchanger or compound 48/80 which inhibits calmodulin, also prevent the expression of the nerve growth factor receptor gene during Wallerian degeneration in vitro. Treatment of the cervical sympathetic trunk with Bepridil leads to loss of the nerve growth factor receptor immunoreactivity which is normally present. The results indicate that Ca2+ may play a role in the expression of the nerve growth factor receptor gene during Wallerian degeneration and provide some indication that this effect may be directly on the Schwann cell rather than operating indirectly via the axon.

Neurones in autonomic ganglia of normal horses contain phosphorylated neurofilaments.

Neurofilaments (NF) are composed of three polypeptides of differing molecular size, termed NF-L, NF-M and NF-H. The NF-H and, to a lesser degree, NF-M components are phosphorylated. In the majority of normal neurones, the location of phosphorylated NF is confined to neuronal processes, particularly the axon, and excluded from the perikaryon. Cell bodies of autonomic neurones of the rat do not contain phosphorylated NF. In many disease states, phosphorylated NF accumulate in the neuronal cell body and therefore in most circumstances their presence indicates abnormality. This paper reports that in at least two autonomic ganglia of normal horses (stellate and coeliomesenteric) the vast majority of neuronal perikarya immunostain strongly for phosphorylated NF. Pretreatment with alkaline phosphatase abolishes staining.

Primary demyelination induced by exposure to tellurium alters Schwann cell gene expression: a model for intracellular targeting of NGF receptor.

Exposure of developing rats to tellurium results in a highly synchronous segmental demyelination of peripheral nerves with sparing of axons; this demyelination is followed closely by a period of rapid remyelination. Demyelination occurs subsequent to a tellurium-induced block in the synthesis of cholesterol, the major myelin lipid. We utilized the techniques of Northern blotting, in situ hybridization, and immunocytochemistry to examine temporal alterations in Schwann cell gene expression related to demyelination and remyelination. Tellurium-induced demyelination is associated with downregulation of myelin protein expression and a corresponding upregulation of NGF receptor (NGF-R) and glial fibrillary acidic protein (GFAP) expression. Steady-state mRNA levels (expressed on a "per nerve" basis) for P0, the major myelin protein, were decreased by about 50% after 5 d of tellurium exposure, while levels of mRNA for NGF-R and GFAP were markedly increased (about 15-fold). In situ hybridization of teased fibers suggested that the increase in steady-state mRNA levels for NGF-R was primarily associated with demyelinated internodes and not with adjacent unaffected internodes. Although P0 message was almost totally absent from demyelinating internodes, it was also reduced in normal-appearing internodes as well. This suggests that limiting the supply of a required membrane component (cholesterol) may lead to partial downregulation of myelin gene expression in all myelinating Schwann cells. In partially demyelinated internodes, NGF-R and GFAP immunofluorescence appeared largely confined to the demyelinated regions. This suggests specific targeting of these proteins to local areas of the Schwann cell where there is myelin loss. These results demonstrate that demyelination is associated with reversion of the affected Schwann cells to a precursor cell phenotype. Because axons remain intact, our results suggest that these changes in Schwann cell gene expression do not require input from a degenerating axon, but instead may depend on whether concerted synthesis of myelin is occurring.

Expression of P0 mRNA in myelinating Schwann cells is related to fibre size.

This study examines whether there is a relationship between the abundance of expression for P0 mRNA in myelinated Schwann cells and fibre diameter. Individual teased sciatic nerve fibres from young adult rats were hybridized with radiolabelled probe for P0 mRNA which is expressed in the perinuclear cytoplasm of the mid-internode. Signal intensity was measured as optical density of the developed autoradiograms. A highly significant positive linear correlation was present between signal intensity and fibre diameter. In a companion study, individual fibres were mounted in Araldite resin and transversely serially sectioned at 4 microns for autoradiography. Grain densities were determined for fibres of different diameters. Again, larger diameter fibres were associated with higher grain densities. The results indicate that the abundance of P0 mRNA expressed by a myelin-producing Schwann cell is related to fibre diameter with axonal size probably being the critical determinant. Axons may regulate P0 expression through the number of signalling molecules exposed on or released from the axolemma.

Expression of myelin protein genes in Schwann cells.

The expression of myelin protein genes in Schwann cells has been studied in situ hybridization. 35S-UTP-labelled, antisense and sense RNA probes to the major protein Po, myelin basic protein (MBP), myelin-associated glycoprotein (MAG) and proteolipid protein (PLP) were employed with paraffin-embedded sections, teased fibres and dissociated Schwann cells from sciatic nerves of rats. Teased fibres were also prepared from cervical sympathetic trunks. Po mRNA was strongly expressed in the mid-internodal perinuclear area of Schwann cell cytoplasm. The degree of signal appeared to be related to fibre size. MBP mRNA showed a diffuse pattern along the Schwann cell internode with a marked increase in grains at the paranodal cytoplasm, particularly in larger fibres. This distribution suggests that the paranodal area is a major site of insertion of MBP into myelin membrane. The expression of MAG and PLP mRNA was markedly lower than Po and MBP. Both mRNAs were localized in the perinuclear cytoplasm and showed a dependence on fibre size. No significant signal was present in Schwann cells associated with unmyelinated axons. In addition to providing data on the cellular expression of myelin protein genes, these studies have shown that teased fibres are invaluable in allowing the localization of low abundance mRNAs.

The distribution of MAG in association with the axonal lesions of canine progressive axonopathy.

The distribution of myelin-associated glycoprotein (MAG) was examined by immunocytochemistry in the spinal cord, accessory cuneate nucleus and lumbar ventral nerve roots of dogs affected by progressive axonopathy. These areas were chosen because of the frequency of spheroids and the associated changes in the myelin sheath, including vacuolation, demyelination, remyelination and accumulation of a granular, amorphous material within the sheath. Normal animals demonstrated the expected distribution of MAG; periaxonal and associated with uncompacted membrane such as Schmidt-Lanterman incisures. The majority of early axonal spheroids were surrounded by a MAG-positive zone but in the larger swellings and longer duration cases this was sometimes absent in places even though the axon was associated with Schwann cell processes. Axons in vacuolated fibres were commonly surrounded by a single adaxonal process of Schwann cell and normal periaxonal space. This was immunoreactive for MAG but in situations where the process was incomplete or the space distorted, staining was absent. The granular material failed to stain for MAG. Distorted Schmidt-Lanterman incisures, a feature of the advanced disease, were strongly positive. In the CNS, spheroids without myelin sheaths or unassociated with oligodendroglial processes were negative for periaxonal MAG. The study confirms the localization of MAG at the periaxonal space. It also raises the question of how the distribution of periaxonal MAG is affected by axonal swelling with a consequent increase in axonal surface area.

Progressive axonopathy: an inherited neuropathy of boxer dogs. An immunocytochemical study of the axonal cytoskeleton.

The distribution of the major axonal cytoskeletal proteins has been determined in lumbar ventral roots and spinal cord of dogs with progressive axonopathy, an inherited neuropathy of boxer dogs. The three neurofilament proteins, and beta-tubulin, actin and fodrin were localized using immunocytochemistry. The majority of swollen axons in the nerve roots contained excessive, disorientated neurofilaments. In about 5% of such fibres the peripheral filaments in the axoplasm were orientated circumferentially and such zones were deficient in tubulin. Many, but not all, spheroids contained increased amounts of actin, often with internal areas of more intense staining. Similar findings were present in axonal swellings in the CNS, although their contents were more variable. The distribution of axonal fodrin in CNS and PNS appeared unaltered. The perikarya of many motor neurons in the spinal cord and brain stem contained phosphorylated neurofilaments. The results support previous suggestions that defects in slow axonal transport are involved in the pathogenesis of this disease.

Progressive axonopathy: an inherited neuropathy of boxer dogs. Quantitative and morphometric analysis of the peripheral nerve lesion.

Previous studies have described and illustrated the lesions in the peripheral nerves in progressive axonopathy, an inherited neuropathy of Boxer dogs. The present paper assesses these changes using quantitative techniques. Cervical and lumbar nerve roots and tibial, phrenic and medial cutaneous radial nerves have been studied in affected and age-matched normal dogs aged 2 months to 3 years. The dorsal and ventral nerve roots, and to a lesser extent the proximal nerves, contain a proportion of swollen myelinated axons whereas in the middle and distal nerves the larger diameter fibres fail to develop to their expected maximum calibre. The unmyelinated axons remain the same size as those in normal dogs. Myelin sheath changes, with attenuation or loss of the sheath and/or remyelination, become increasingly prevalent through the course of the disease, always maintaining a proximal to distal decrease in their frequency. Quantification indicates that, particularly in the ventral roots, many axons have disproportionately thin sheaths with shortened internodes. Axonal degeneration and regeneration increase in frequency in the distal nerves as the disease progresses. The cervical ventral roots prove an exception in that they contain large numbers of regenerating clusters at most stages. It is suggested that in progressive axonopathy an axonal transport failure may occur in the roots leading to the axonal swellings, as a result of which a developmental hypoplasia occurs in the more distal, larger diameter fibres. The prominent, but unevenly distributed, myelin sheath changes indicate a severe disturbance in axon-sheath cell inter-relationships.

Effect of surfactant poloxalene 2930 on food intake, lipid absorption, and serum cholesterol in rats.

Effect of hydrophobic surfactant, poloxalene 2930, on lipid absorption was studied in rats. Under acute conditions with surfactant infused intraduodenally with a lipid meal absorbed lipid accumulated abnormally in the enterocytes. This effect was quickly reversed after terminating treatment. Long-term administration of poloxalene given in semipurified diets resulted in changes in food intake, weight gain, fecal fat output, and serum cholesterol concentrations. The composition of the diet used as the vehicle for administration had a considerable effect on these results. When semipurified diets were used, food intake and weight gain were greatest when the dietary fat content was at the highest level. When the surfactant was given in ground chow, food intake was not affected and weight gain was only slightly, but significantly, less than the controls as a result of mild fat malabsorption. It is concluded that poloxalene 2930 affects lipid absorption, food intake, and serum cholesterol concentration but that results of this treatment are considerably affected by dietary factors.

Hydrophobic surfactant treatment prevents atherosclerosis in the rabbit.

The hypocholesterolemic effect of the hydrophobic surfactant, poloxalene 2930, was studied in the rabbit to determine whether this agent prevents experimentally produced atherosclerosis. Male rabbits were divided into four groups and fed a control diet (group A) or an atherogenic diet (groups B, C, and D) for 10 wk. Diets of groups C and D were supplemented with 0.5 and 1% poloxalene 2930, respectively. Animals in group B developed significantly greater levels of cholesterol in the serum and aorta compared with group A. Addition of poloxalene 2930 to the diets of groups C and D prevented significant elevations in cholesterol concentrations of both serum and aorta compared with group B with values for group D being essentially similar to those observed in group A. Groups C and D also had significant increases of fecal excretion of both neutral fat and neutral steroids as compared with either groups A or B. There were no atherosclerotic lesions of the aortas from group D. Aortas from rabbits in group B had numerous atheromatous plaques while one rabbit each from groups A and C had several very small atheromatous lesions. These results demonstrate that poloxalene 2930 reduces the rise of serum cholesterol in rabbits in response to an atherogenic diet and prevents the development of atherosclerosis. This hypocholesterolemic effect is likely mediated by the effect of this surfactant on the small intestine.

Desaturation of endogenous and exogenous palmitate in lung tissue in vitro.

lung slices from rats fed a fat-free diet supplemented with safflower oil (control) or tripalmitoylglycerol (essential fatty acid [EFA]-deficient) were incubated with [14C]acetate, [14C]palmitate, or [14C]stearate. Of the 14C recovered in phospholipids after incubation with [14C]acetate, more than 87% was in 16-carbon fatty acids. Desaturation, as assayed by the percentage of radioactivity in monoenoates in phospholipid fatty acids, was generally double in EFA-deficient slices compared to control slices, regardless of substrate. Desaturation was significantly greater in slices incubated with with acetate or octanoate compared to palmitate, indicating that endogenously synthesized palmitate was desaturated more actively than that derived from an exogenous source.