Idiopathic intracranial hypertension associated with iron deficiency anaemia: a lesson for management.

AIM: To document the causal association of iron deficiency anaemia (IDA) and intracranial hypertension (IH). METHODS: A consecutive case note review of patients with a clinical diagnosis of idiopathic intracranial hypertension (IIH) and anaemia presenting to a tertiary referral unit over a 2.5-year period. Demographics, aetiology and clinical details were recorded and analysed. RESULTS: Eight cases were identified from 77 new cases presenting with IIH. All 8 had documented microcytic anaemia with clinical evidence of raised intracranial pressure. There was no evidence of venous sinus thrombosis on MRI and MR venography in 7 subjects and on repeated CT venography in 1. On correction of anaemia alone, 7 cases resolved. One patient with severe progressive visual loss underwent ventriculoperitoneal shunt in addition to treatment of anaemia, with good outcome. The incidence of this association is 10.3%. CONCLUSION: These cases present an association between IDA and IH, in the absence of cerebral sinus thrombosis. As a clinically significant proportion of cases presenting with signs of IIH have IDA, we recommend all patients presenting with IIH have full blood counts and if they are found to be anaemic, they should be treated appropriately.

Elevated cerebrospinal fluid (CSF) leptin in idiopathic intracranial hypertension (IIH): evidence for hypothalamic leptin resistance?

OBJECTIVE: The aetiology of idiopathic intracranial hypertension (IIH) is not known, but its association with obesity is well-recognized. Recent studies have linked obesity with abnormalities in circulating inflammatory and adiposity related cytokines. The aim of this study was to characterize adipokine and inflammatory cytokine profiles in IIH. DESIGN: Paired serum and cerebrospinal fluid (CSF) specimens were collected from 26 patients with IIH and compared to 62 control subjects. Samples were analysed for leptin, resistin, adiponectin, insulin, IL-1beta, IL-6, IL-8 (CXCL8), TNFalpha, MCP-1 (CCL2), hepatocyte growth factor, nerve growth factor and PAI-1 using multiplex bead immunoassays. RESULTS: CSF leptin was significantly higher in patients with IIH (P = 0.001) compared to controls after correction for age, gender and body mass index (BMI). In the control population, BMI correlated with serum leptin (r = 0.34; P = 0.007) and CSF leptin (r = 0.51; P < 0.0001), but this was not the case for the IIH population. Profiles of other inflammatory cytokines and adipokines did not differ between IIH patients and controls once anthropometric factors had been accounted for. CONCLUSIONS: IIH was characterized by significantly elevated CSF leptin levels which did not correlate with BMI. We suggest that CSF leptin may be important in the pathophysiology of IIH and that obesity in IIH may occur as a result of hypothalamic leptin resistance.

Ciliary neurotrophic factor protects retinal ganglion cells from axotomy-induced apoptosis via modulation of retinal glia in vivo.

Adenoviral-mediated transfer of ciliary neurotrophic factor (CNTF) to the retina rescued retinal ganglion cells (RGCs) from axotomy-induced apoptosis, presumably via activation of the high affinity CNTF receptor alpha (CNTFRalpha) expressed on RGCs. CNTF can also activate astrocytes, via its low affinity leukemia inhibitory receptor beta expressed on mature astrocytes, suggesting that CNTF may also protect injured neurons indirectly by modulating glia. Adenoviral-mediated overexpression of CNTF in normal and axotomized rat retinas was examined to determine if it could increase the expression of several glial markers previously demonstrated to have a neuroprotective function in the injured brain and retina. Using Western blotting, the expression of glial fibrillary acid protein (GFAP), glutamate/aspartate transporter-1 (GLAST-1), glutamine synthetase (GS), and connexin 43 (Cx43) was examined 7 days after intravitreal injections of Ad.CNTF or control Ad.LacZ. Compared to controls, intravitreal injection of Ad.CNTF led to significant changes in the expression of CNTFRalpha, pSTAT(3), GFAP, GLAST, GS, and Cx43 in normal and axotomized retinas. Taken together, these results suggest that the neuroprotective effects of CNTF may result from a shift of retinal glia cells to a more neuroprotective phenotype. Moreover, the modulation of astrocytes may buffer high concentrations of glutamate that have been shown to contribute to the death of RGCs after optic nerve transection.

Effects of adenoviral-mediated gene transfer of interleukin-10, interleukin-4, and transforming growth factor-beta on the survival of axotomized retinal ganglion cells.

Nitric oxide, synthesized by reactive microglia and astrocytes has been implicated in promoting neuronal degeneration observed in many diseases and insults of the central nervous system. We have recently shown that inducible nitric oxide synthase is expressed by retinal glial cells following optic nerve transection and that inhibition of nitric oxide synthesis enhances the survival of injured retinal ganglion cells. Anti-inflammatory cytokines including interleukin-10 (IL-10), interleukin-4 (IL-4), and transforming growth factor-beta (TGF-beta) have been shown to prevent inducible nitric oxide synthase expression, and inhibit nitric oxide synthesis by microglia and astrocytes in culture. In the present study, we examined the effects of adenoviral mediated gene transfer of anti-inflammatory cytokines on the survival of axotomized retinal ganglion cells. Intraocular administration of adenoviral vectors encoding interleukin-10 (Ad.IL-10) and interleukin-4 (Ad.IL-4) enhanced the survival of axotomized retinal ganglion cells at 14 days after axotomy. Adenoviral vectors encoding TGF-beta (Ad.TGF-beta) had no effect on retinal ganglion cell survival. Separate animals were pretreated by injection of Ad.IL-10 or Ad.IL-4 into the superior colliculus (s.c.), the major target of ganglion cells, 7 days prior to axotomy. S.c. administration of Ad.IL-10 or Ad.IL-4 significantly increased ganglion cell survival compared with intraocular injection. IL-10 and IL-4 gene transfer also reduced the density of infiltrating ED1 positive monocytes in the nerve fiber layer at 14 days postaxotomy. Ad.TGF-beta increased the density of ED1 positive monocytes infiltrating the nerve fiber layer after axotomy. Vectors encoding IL-10 or IL-4 also decreased nitrotyrosine immunoreactivity in the inner retina at 7 days postaxotomy, suggesting that these cytokines protect retinal ganglion cells from peroxynitrite formation that results from nitric oxide synthesis by activated glial cells. The present study has implications for the treatment of CNS injury and diseases that involve reactive microglia and astrocytes. Our results suggest that interleukin-10 and interleukin-4 may help prevent neurodegeneration caused by the activation of glial cells after CNS injury.

Expression of the 40 kDa catecholamine regulated protein in the normal and injured rat retina.

Catecholamine regulated protein 40 (CRP40) has been shown to be expressed in the central nervous system (CNS) of several mammalian species where it may function in a similar manner to members of the heat shock protein (HSP) family. Immunohistochemical and immunoblotting techniques were utilized to investigate whether CRP40 is expressed in normal rat retinas. In addition, changes in CRP40 expression were studied following optic nerve transection. The immunohistochemical results showed that CRP40 is expressed in the normal rat retina. The protein was found to be highly expressed in the ganglion cell layer (GCL), the inner nuclear layer (INL) and the outer plexiform layer (OPL). In addition, a low level of CRP40 was found in the inner plexiform layer (IPL), and in the inner segment layer (ISL). No expression was found in the outer nuclear layer (ONL) of normal rat retina. The immunoblotting results show that CRP40 expression decreased in a time-dependent fashion after the optic nerve transection. This decrease indicates that the expression of CRP40 is dependent on the neuron's normal physiological state and that it plays an important function in physiological and pathological conditions in the retina.

Transection of dysmyelinated optic nerve axons in adult rats lacking myelin basic protein.

Injury to myelin or oligodendrocytes may manifest as dysmyelinating or demyelinating conditions of the CNS. Previous studies using dysmyelinated animal models (myelin basic protein mutants) suggest possible axonal dysfunction with complete loss of myelin. In this present study, we evaluated retinal ganglion cell survival after axotomy in MBP mutants to determine if prolonged dysmyelination of CNS axons exerted a detrimental effect on neuronal survival. We demonstrated that the survival of retinal ganglion cells with dysmyelinated axons is identical to retinal ganglion cells with myelinated axons after survival times up to 180 days. In myelin diseases where axon transection is a consistent consequence of demyelination resulting in progressive neurological deterioration, the absence of myelin does not accelerate neuronal death.

Nitric oxide synthase inhibition delays axonal degeneration and promotes the survival of axotomized retinal ganglion cells.

Nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been implicated in neuronal cytotoxicity following trauma to the central nervous system. The aim of the present study was to examine the role of NO in mediating axotomy-induced retinal ganglion cell (RGC) death. We observed increases in iNOS expression by microglia and Müller cells in the retina after optic nerve transection. This was paralleled by the induced expression of constitutive NOS (cNOS) in RGCs which do not normally express this enzyme. In order to determine if NO is cytotoxic to axotomized RGCs, the nonspecific NOS inhibitors Nomega-nitro-L-arginine (NOLA) or N-nitro-L-arginine methyl ester (L-NAME) were delivered to the vitreous chamber by intraocular injections. Both NOLA and L-NAME significantly enhanced RGC survival at 7, 10, and 14 days postaxotomy. The separate contributions of iNOS and cNOS to RGC degeneration were examined with intraocular injections of the specific iNOS inhibitor L-N(6)-(I-iminoethyl)lysine hydrochloride or the specific cNOS inhibitor L-thiocitrulline. Our results suggest that cNOS plays a greater role in RGC degeneration than iNOS. In addition to enhancing RGC survival, NOS inhibitors delayed the retrograde degeneration of RGC axons after axotomy. We conclude that NO synthesized by retinal iNOS and cNOS plays a major role in RGC death and retrograde axonal degeneration following axotomy.

Effects of GDNF on retinal ganglion cell survival following axotomy.

Recent evidence suggests that approximately 90% of retinal ganglion cells (RGCs) die by the process of apoptosis within 14 days of optic nerve transection. RGCs begin to disappear from the retina between 5 and 7 days postaxotomy when the highest percentage of RGCs show characteristics typical of apoptosis. A single intraocular injection of glial cell-line derived neurotrophic factor (GDNF) given at the time of axotomy resulted in a delay in the initiation of RGC death and increased the densities of surviving RGCs at 7, 10 and 14 days postaxotomy. The mean RGC densities in GDNF treated retinas at 7 (2381 +/- 144), 10 (1561 +/- 117) and 14 (1123 +/- 116) days postaxotomy were significantly higher than that of controls (1835 +/- 82, 835 +/- 272 and 485 +/- 39, respectively). The loss of RGCs was paralleled by increases in TUNEL positive staining in control retinas and a lower percentage of TUNEL positive cells in GDNF treated retinas at 5, 7 and 10 days postaxotomy. These results suggest that GDNF is capable of promoting RGC survival following injury, possibly by interfering with an essential step in apoptosis.

Localization of gap junctions and tracer coupling in retinal Müller cells.

Physiological studies have demonstrated the existence of direct intercellular communication, presumably mediated by gap junctions, both between neurons and between glial cells in the vertebrate retina. We localized gap junctions in the retinas of rat, goldfish, and mudpuppy by using antisera directed against proteins that make up the connexon channels in two tissues from which connexins have been isolated: liver (connexin 32; CX32) and heart (connexin 43; CX43). Although the antiserum against CX32 stained liver gap junctions, it did not reveal any staining in rat or goldfish retina. The antiserum against CX43 stained gap junctions associated with the intercalated disk in rat heart and also stained gap junctions between pigment epithelium cells in rat, goldfish, and mudpuppy retina. Anti-CX43 also stained gap junctions between Müller cells in goldfish and mudpuppy retina but not in rat retina. Intracellular injections of the tracer Neurobiotin into Müller cells in the mudpuppy retina revealed that these glial cells are extensively tracer coupled. Staining with the tracer formed a syncytium of thin processes surrounding every neuron from the outer limiting membrane to the inner limiting membrane. Confocal microscopy demonstrated that the Müller cells were in close apposition with one another at every level of the retina. However, CX43 immunoreactivity was heaviest at the outer limiting membrane, where the apical processes of Müller cells are located. Some anti-CX43 staining was observed at the level of the outer nuclear layer and the inner plexiform layer but not in the ganglion cell layer or at the Müller cell end feet forming the inner limiting membrane.

Intercellular metabolic coupling in canine colon musculature.

Intercellular communication within the musculature of the canine colon was studied by examining the results of neurobiotin diffusion after injection of the tracer into smooth muscle cells at different locations within the muscle layer. Circular muscle at the submucosal surface, circular muscle adjacent to the myenteric plexus, and longitudinal muscle demonstrated different degrees of time-dependent tracer spread. At the submucosal surface, tracer spread was rapid, extensive, and unimpeded by connective tissue septa. At the myenteric side, tracer spread was also extensive but was much slower and confined to bundles of cells bordered by septa. In contrast to previous studies that suggest an absence of gap junctions at the myenteric side of the circular muscle, the neurobiotin spread indicates full metabolic coupling of all circular smooth muscle cells. Furthermore, in contrast to the belief that longitudinal muscle is completely devoid of gap junctions, tracer spread occurred between cells in this layer, although neurobiotin diffusion was very limited, nonuniform, and slow. In each area of the musculature studied, tracer spread was inhibited by octanol. When very long injection and wait times were implemented at the submucosal surface of the circular muscle, neurobiotin was observed to cross septa through the network of interstitial cells of Cajal, indicating that it is this network that provides communication between lamellae.

Evidence for D2 receptor regulation of dopamine release in the goldfish retina.

The possible existence of a dopamine D2 receptor-mediated regulation of dopamine release was investigated in the goldfish retina. Isolated retinas were preloaded with [3H]dopamine and superfused with D2 dopamine receptor agonists or antagonists to determine if there was an effect on [3H]dopamine release. The D2 receptor antagonist sulpiride increased both baseline [3H]-dopamine release and [3H]dopamine release induced by an increase in extracellular potassium concentration. The D2 receptor agonists LY-171555 and RU-24213 did not reduce baseline [3H]dopamine release but completely inhibited [3H]dopamine release induced by an increase in [K+]o. This action of the D2 agonists was blocked by sulpiride. These studies demonstrate the existence of D2 receptor, possibly autoreceptor, regulation of dopamine release in the teleost retina.

Effects of norepinephrine on [3H]dopamine release and horizontal cell receptive-field size in the goldfish retina.

Norepinephrine increased the release of pre-loaded [3H]dopamine from goldfish retinas. Pharmacological studies suggested that the norepinephrine-induced [3H]dopamine release was due to an exchange mechanism between norepinephrine and pre-loaded [3H]dopamine. Norepinephrine also depolarized and reduced the receptive-field size of horizontal cells in goldfish retinas. The action of norepinephrine on horizontal cells was probably not due to the release of endogenous dopamine because the effect of norepinephrine was not abolished in retinas in which all dopaminergic neurons had been destroyed by prior treatment with 6-hydroxydopamine. The pharmacology of the effect of norepinephrine on horizontal cells suggested that it was due to an agonist action of norepinephrine acting at horizontal cell dopamine receptors. It is still unclear whether endogenous norepinephrine is a regulator of dopamine release in the fish retina. Consequently, the function of the putative norepinephrine-containing amacrine cells of the fish retina remains to be elucidated.

A new type of interplexiform cell in the goldfish retina is PNMT-immunoreactive.

A new cell type immunoreactive for phenylethanolamine N-methyltransferase, the synthesizing enzyme for epinephrine, has been identified in the goldfish retina. The somata of the immunoreactive cells were located in the proximal inner nuclear layer and immunoreactive processes were located in both the inner and outer plexiform layers, suggesting that this cell may be an interplexiform cell. Confocal microscopy was used to establish that the putative adrenergic neurites in the outer plexiform layer were located between the somata of horizontal cells and photoreceptor cell synaptic terminals. Intracellular recordings from horizontal cells demonstrated that epinephrine had an effect on horizontal cells, but that the action of epinephrine was consistent with an effect on dopamine receptors, rather than adrenergic receptors. The function of the putative 'I3' interplexiform cell therefore remains unclear, possibly modulating horizontal cell function with a transmitter other than epinephrine or affecting photoreceptors or the retinal pigment epithelium rather than horizontal cells.

Neuromodulation of pigment movement in the RPE of normal and 6-OHDA-lesioned goldfish retinas.

The role of dopamine as the endogenous signal-initiating light-dependent changes in the distribution of pigment granules in goldfish retinal pigment epithelium was investigated. In normal retinas, light adaptation resulted in the dispersion of pigment granules. This effect of light was mimicked by the intraocular injection of dopamine or serotonin, which is thought to increase endogenous dopamine release, into dark-adapted eyes. The effect of light, dopamine, or serotonin on dark-adapted retinas was blocked by the dopamine receptor antagonists haloperidol and sulpiride. However, lesioning the endogenous source of retinal dopamine, by prior intraocular injection of 6-hydroxydopamine (6-OHDA), did not block the dispersion of pigment granules in light-adapted retinas. No significant differences in pigment dispersion were noted between unlesioned and lesioned light- or dark-adapted retinas. However, the effect of light on pigment dispersion was no longer blocked by haloperidol or sulpiride in 6-OHDA lesioned animals. Dopamine and serotonin mimicked the effect of light when injected into lesioned dark-adapted eyes, but their effects were also not blocked by haloperidol or sulpiride. These results suggest that dopamine, acting on D2 receptors, is sufficient to induce pigment migration in unlesioned animals. In 6-OHDA-lesioned animals, however, pigment migration is mediated by a receptor mechanism other than D2.

Background illumination reduces horizontal cell receptive-field size in both normal and 6-hydroxydopamine-lesioned goldfish retinas.

The effect of background illumination on horizontal cell receptive-field size and dye coupling was investigated in isolated superfused goldfish retinas. Background illumination reduced both horizontal cell receptive-field size and dye coupling. The effect of light on horizontal cell receptive-field size was mimicked by treating the retina with 20 microM dopamine. To test the hypothesis that the effects of light were due to endogenous dopamine release, the effect of light was studied in goldfish retinas in which dopaminergic interplexiform cells were lesioned using 6-hydroxydopamine treatment. In lesioned retinas, background illumination reduced both horizontal cell receptive-field size and dye coupling. Furthermore, the effect of background illumination on unlesioned animals could not be blocked by prior treatment with the D1 dopamine receptor antagonist SCH-23390. These results suggest that, in goldfish retina, dopamine release is not the only mechanism by which horizontal cell receptive-field size could be reduced by light.

Enkephalinergic modulation of the dopamine system in the turtle retina.

One subpopulation of amacrine interneurons in the turtle retina was shown to contain met-enkephalin by means of immunocytochemistry, and another was demonstrated to have a high-affinity uptake system for [3H]-dopamine by means of autoradiography. Although the amacrine soma size, density, and distribution of their neurites in IPL substrata was similar in retinas in which met-enkephalin and dopamine were localized, combined light microscope immunocytochemistry-autoradiography demonstrated that these two neurotransmitter systems did not coexist in the same cells. Because the two amacrine cell subtypes ramify in the same IPL substrata, neuronal interaction between them is possible. Release experiments showed that the potassium-induced release of [3H]-dopamine from the superfused turtle retina was reduced by 40% when enkephalin was added to the superfusate. The inhibition of [3H]-dopamine release could be blocked by the addition of naloxone. The addition of enkephalin had no effect of the potassium-induced release of [3H]-GABA from the superfused retina. These findings suggest that an enkephalinergic modulation of the dopaminergic amacrine cell system exists in the turtle retina.

Gap junction particle density of horizontal cells in goldfish retinas lesioned with 6-OHDA.

The I1 dopaminergic interplexiform cells of the fish retina are believed to modulate horizontal cell coupling by increasing gap junction resistance. Dopamine also modulates the morphology of horizontal cell gap junctions and mimics the effects of light adaptation. To determine whether the light-dependent changes in gap junction morphology are due to endogenous dopamine release, horizontal cell gap junctions were studied in goldfish retinas lacking dopaminergic neurons. Dopaminergic interplexiform cells were destroyed by intraocular injections of 6-hydroxydopamine in both eyes. After lesioning, fish were treated in one of four ways: (1) light-adapted, (2) dark-adapted (1 hour), (3) light-adapted and given an intraocular injection of dopamine, or (4) dark-adapted (1 hour) and injected with dopamine. The effectiveness of lesioning was evaluated by autoradiographic detection of [3H]-dopamine uptake in the retina of one eye. Retinas in which lesioning of the contralateral eye was deemed effective were processed for freeze-fracture electron microscopy and the particle density of horizontal cell gap junctions determined. Lesioned retinas, whether light- or dark-adapted, had elevated horizontal cell soma gap junction particle densities compared to lesioned retinas treated with dopamine. These results demonstrate that high soma gap junction particle densities can be correlated with the absence of dopamine and low densities associated with the presence of dopamine. The differences in gap junction particle density between lesioned and lesioned + dopamine-treatment were similar to differences between nonlesioned dark-adapted (1 hour) and light-adapted retinas, respectively. Therefore, the particle density of light- and dark-adapted soma gap junctions suggests a greater release of dopamine in light-adapted fish than in 1 hour dark-adapted fish.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopaminergic regulation of horizontal cell gap junction particle density in goldfish retina.

Light- or dark-adapted goldfish (Carassius auratus) retinas were treated with dopamine, which is believed to uncouple horizontal cells via D1 receptors, or with the dopamine antagonist haloperidol. Aldehyde-fixed retinas were freeze-fractured and the replicas examined by electron microscopy to identify horizontal gap junctions. The density (number per micron2) of intra-membrane particles of horizontal cell soma gap junctions was significantly lower in light-adapted and dopamine-treated retinas than in dark-adapted and haloperidol-treated retinas. There was no statistically significant difference between gap junction particles densities in (I) light-adapted (untreated) and in dopamine-treated (light- or dark-adapted) retinas, or between (II) dark-adapted (untreated) and haloperidol-treated (light- or dark-adapted). These results suggest that the uncoupling of horizontal cell somas by dopamine is accompanied by a decrease in gap junction particle density and that there is a greater release of dopamine during light-adaptation than dark-adaptation. Unlike horizontal cell somas, horizontal cell axon terminals did not show consistent changes in gap junction particle density with light- or dark-adaptation. Although the data suggests that there may be a reduction in axon terminal gap junction particle density with dopamine treatment, this effect is not reversible with haloperidol treatment. Our results suggest that the regulation of gap junctions may differ at two sites within the same cell.

Immunocytochemical and autoradiographic localization of GABAergic neurons in the goldfish retina.

The putative neurotransmitter gamma-aminobutyric acid (GABA) was localized in goldfish retina by using an antiserum directed against GABA itself. The same types of cells were stained with this antibody as were labelled with an antiserum directed against the synthesizing enzyme for GABA, glutamic acid decarboxylase. Stained neurites of these cells were located throughout the inner plexiform layer (IPL) but staining was more intense in the proximal IPL. The GABA-immunoreactive staining could be reduced or completely abolished by preabsorbing the primary antibody with GABA. Uptake of [3H]-GABA or the GABA agonist [3H]-muscimol was localized in GABA-stained retinas using light microscope autoradiography. These experiments demonstrated that all types of GABA-immunoreactive amacrine cells had high-affinity uptake mechanisms for both [3H]-GABA and -muscimol. Thirty percent of proximal inner nuclear layer (INL) and some cells in the ganglion cell layer (GCL) were labelled by all three GABAergic markers. Most GABA-immunoreactive amacrine cells were lightly labelled due to [3H]-GABA uptake but a few amacrines (Ab) were heavily labelled. These findings demonstrate that the autoradiographic localization of [3H]-GABA or [3H]-muscimol uptake and the immunocytochemical localization of GAD or GABA are appropriate methods for localizing GABAergic neurons in the retina. Few GABA-immunoreactive amacrine cells accumulated the putative amino acid transmitter [3H]-glycine, verifying that the goldfish retina contains distinct subpopulations of glycinergic and GABAergic amacrine cells.