Nancy E.J. Berman

Overexpression of Monocyte Chemoattractant Protein 1 in the Brain Exacerbates Ischemic Brain Injury and Is Associated With Recruitment of Inflammatory Cells

Brain cells produce cytokines and chemokines during the inflammatory process after stroke both in animal models and in patients. Monocyte chemoattractant protein 1 (MCP-1), one of the proinflammatory chemokines, can attract monocytes to the tissue where MCP-1 is overexpressed. However, the role of MCP-1 elevation in stroke has not been explored in detail. The authors hypothesized that elevated MCP-1 levels would lead to increased influx of monocytes and increased brain infarction size in stroke induced by middle cerebral artery occlusion with partial reperfusion. There were no differences in blood pressure, blood flow, or vascular architecture between wild-type mice and transgenic MBP-JE mice. Twenty-four to 48 hours after middle cerebral artery occlusion, brain infarction volumes after ischemia were significantly larger in MBP-JE mice than in wild-type controls and were accompanied by increased local transmigration and perivascular accumulation of macrophages and neutrophils. These results indicate that MCP-1 can contribute to inflammatory injury in stroke.

Exacerbated glial response in the aged mouse hippocampus following controlled cortical impact injury

Old age is associated with enhanced susceptibility to and poor recovery from brain injury. An exacerbated microglial and astrocyte response to brain injury might be involved in poor outcomes observed in the elderly. The present study was therefore designed to quantitate the expression of markers of microglia and astrocyte activation using real-time RT-PCR, immunoblot and immunohistochemical analysis in aging brain in response to brain injury. We examined the hippocampus, a region that undergoes secondary neuron death, in aged (21-24 months) and adult (5-6 months) mice following controlled cortical impact (CCI) injury to the sensorimotor cortex. Basal mRNA expression of CD11b and Iba1, markers of activated microglia, was higher in aged hippocampus as compared to the adult. The mRNA expression of microglial markers increased and reached maximum 3 days post-injury in both adult and aged mice, but was higher in the aged mice at all time points studied, and in the aged mice the return to baseline levels was delayed. Basal mRNA expression of GFAP and S100B, markers of activated astrocytes, was higher in aged mice. Both markers increased and reached maximum 7 days post-injury. The mRNA expression of astrocyte markers returned to near basal levels rapidly after injury in the adult mice, whereas again in the aged mice return to baseline was delayed. Immunochemical analysis using Iba1 and GFAP antibodies indicated accentuated glial responses in the aged hippocampus after injury. The pronounced and prolonged activation of microglia and astrocytes in hippocampus may contribute to worse cognitive outcomes in the elderly following TBI.

Selective chemokine mRNA expression following brain injury

Injury in non-neuronal tissues stimulates chemokine expression leading to recruitment of inflammatory cells responsible for orchestration of repair processes. The signals involved in directing repair of damage to the brain are less well understood. We hypothesized that following brain injury, chemokines are expressed and regulate the rate and pattern of inflammatory cell accumulation. The two chemokine subfamilies are alpha(alpha)-chemokines, which primarily function as neutrophil chemoattractants, and the beta(beta)-chemokines, which function primarily as monocyte chemoattractants. We assessed alpha and beta chemokine mRNA expression patterns and leukocyte accumulation following a cerebral cortical lesion. Cortical lesions were produced with and without addition of endotoxin, Escherichia coli lipopolysaccharide (LPS), which stimulates cytokine expression. We studied the expression of the beta-chemokines: monocyte chemoattractant protein (gene product JE; MCP-1/JE), macrophage inflammatory protein-1 alpha and beta (MIP-1alpha and MIP-1beta), and the regulated upon activation normal T expressed and secreted chemokine (RANTES) as well as the alpha-chemokines: interferon-gamma-inducible protein (IP-10) and N51/KC (KC; a murine homologue of MIP-2). Changes in gene expression were analyzed by Northern analysis at different time points following injury. Leukocyte and macrophage densities were analyzed by immunohistochemistry at the same time intervals. All chemokines were elevated following cortical injury/endotoxin. MCP-1 and MIP-1alpha were elevated at 2 h and peaked 6 h, MIP-1beta peaked at 6 h, but declined more rapidly than MCP-1 or MIP-1alpha, and IP-10 peaked at 6 h and showed the most rapid decline. KC was elevated at 1 h, and peaked at 6 h following LPS. RANTES was elevated at 1 h and achieved a plateau level between 6 and 18 h, then declined. In contrast, sterile injuries produced in the absence of endotoxin only induced the mRNA of the beta-chemokine MCP-1, and its expression was delayed compared to the cortical injury/endotoxin group. The presence of chemokine message as early as 1 h indicates that expression of this class of molecules is an early response in the repair process following traumatic brain injury. Macrophage/microglia accumulation occurred more rapidly, activated microglia further from the lesion border, and more cells accumulated in cortical injury/endotoxin than in cortical lesions produced under sterile conditions. Thus, there was a positive correlation between beta-chemokine expression and the number of beta-chemokine responsive cells (i.e. microglia) accumulating in injury sites. This is the first comprehensive study using a panel of chemokine probes and specific marcophage/microglial markers to study in vivo activation of the brain following injury. Our data show that the brain is capable of expression of multiple chemokine genes upon appropriate stimulation (e.g. LPS-treatment). The gradient of microglial activation is consistent with physical damage stimulating release of chemokines that diffuse from the injury site. These data strongly suggest that chemokines are instrumental in the initiation of repair processes following brain injury.

Pathogenic implications of iron accumulation in multiple sclerosis

J. Neurochem. (2012) 120, 7–25.

Alterations in connections of the corpus callosum following convergent and divergent strabismus

The connectivity of the corpus callosum in visual cortical areas 17 and 18 was studied in normal cats, in cats reared with unilateral convergent or divergent surgically-induced strabismus, and in a Siamese cat. The extents of the callosal cell and terminal zones were determined following multiple injections of horseradish peroxidase and tritiated amino-acids into one hemisphere. Following surgically-induced strabismus, abnormally wide callosal cell zones were seen in both the left and the right hemisphere irrespective of the direction of eye misalignment. Abnormally wide callosal terminal zones were seen in the hemisphere ipsilateral to the deviating eye in cats reared with unilateral convergent and divergent strabismus. Abnormally wide callosal zones were seen in cats which had strabismus induced as late as postnatal day 36. In a Siamese cat with a naturally-occurring convergent strabismus, callosal cells had a different distribution and were fewer in number compared to normal cats or cats with surgically induced strabismus. This implies that the abnormal callosal connectivity of Siamese cats is not a simple result of strabismus.

Ovarian steroids regulate neuropeptides in the trigeminal ganglion

Women are more than three times as likely as men to experience migraine headaches and temporomandibular joint pain, and painful episodes are often linked to the menstrual cycle. To understand how hormone levels may influence head and face pain, we assessed expression of pain-associated neuropeptides and estrogen receptor alpha (ERalpha) during the natural estrous cycle in mice. Gene expression was analyzed in the trigeminal ganglia of cycling female mice at proestrus, estrus and diestrus using RT-PCR. Peptide/protein expression in trigeminal neurons was analyzed using immunohistochemistry. ERalpha mRNA was present at all stages and highest at estrus. ERalpha protein was present in the cytoplasm of medium-sized and small trigeminal neurons. ERalpha immunoreactive neurons were most common at diestrus. CGRP and ANP mRNAs did not change across the estrous cycle, while expression of galanin and NPY mRNAs were strongly linked to the estrous cycle. Galanin mRNA levels peaked at proestrus, when expression was 8.7-fold higher than the diestrus levels. Galanin immunoreactivity also peaked at proestrus. At proestrus, 7.5% of trigeminal neurons contained galanin, while at estrus, 6.2% of trigeminal neurons contained galanin, and at diestrus, 4.9% of trigeminal neurons contained galanin. NPY mRNA peaked at estrus, when levels were 4.7-fold higher than at diestrus. Our findings suggest that estrogen receptors in trigeminal neurons modulate nociceptive responses through effects on galanin and NPY. Variations in neuropeptide content in trigeminal neurons across the natural estrous cycle may contribute to increases in painful episodes at particular phases of the menstrual cycle.

Cortical spreading depression and gene regulation: Relevance to migraine

Cortical spreading depression (CSD) may be the underlying mechanism of migraine aura. The role of CSD in initiating a migraine headache remains to be determined, but it might involve specific changes in gene expression in the brain. To examine these changes, four episodes of CSD at 5‐minute intervals were induced in the mouse brain by application of 300mM KCl, and gene expression was examined 2 hours later using cDNA array and reverse transcriptase–polymerase chain reaction. Controls consisted of groups that received anesthesia only, attachment of recording electrodes only, and application of 0.9% NaCl. Of the over 1,180 genes examined in our experiments, those consistently regulated by CSD included vasoactive peptides; the vasodilator atrial natriuretic peptide was induced by CSD, while the vasoconstrictor neuropeptide Y was downregulated. Other genes specifically regulated by CSD were involved in oxidative stress responses (major prion protein, glutathione‐S‐transferase‐5, and apolipoprotein E). L‐type calcium channel mRNA was upregulated. In summary, CSD regulates genes that are intrinsic to its propagation, that identify accompanying vascular responses as a potential source of pain, and that protect against its potential pathological consequences. We believe these observations have strong relevance to the mechanisms of migraine and its outcomes.

A mouse model of sensorimotor controlled cortical impact: Characterization using longitudinal magnetic resonance imaging, behavioral assessments and histology

The present study establishes a new mouse model for traumatic brain injury (TBI), using an electromechanically driven linear motor impactor device to deliver a lateral controlled cortical impact (CCI) injury to the sensorimotor cortex. Lesion cavity size was measured, and inter-animal consistency demonstrated, at 14 days post injury. Qualitative information regarding damage progression over time was obtained by scanning with high field magnetic resonance imaging (MRI) at five time points following injury. Functional impairment and recovery were measured with the Rotarod, gridwalk and cylinder tests, and lesion cavity volume was measured post mortem with thionin-stained tissue sections. The study establishes the reliability of a linear-motor based device for producing repeatable damage in a CCI model, demonstrates the power of longitudinal MRI in studying damage evolution, and confirms that a simple battery of functional tests record sensorimotor impairment and recovery.

Chemical modulation of metallothionein I and III mRNA in mouse brain

Metallothioneins (MTs) are sulfhydryl-rich proteins. MT-I and MT-II are found in all tissues of the body, while MT-III exists only in brain. Regulation of MT-I and MT-III mRNA was studied in brain and liver of control C57BL/6J mice and mice given chemicals known to increase MT-I, namely, lipopolysaccharide (LPS), zinc chloride (Zn), cadmium chloride (Cd), dexamethasone (Dex), ethanol, and kainic acid (KA). Northern blot analysis revealed that MT-I mRNA levels in liver were induced dramatically (12-27-fold over basal levels) by all of the chemicals, while in brain only LPS produced an increase in MT-I mRNA (2-fold). Interestingly, the MT-I inducers, Cd, Dex, ethanol, and KA, down-regulated brain MT-III mRNA levels by approx. 30%. Because brain is such a heterogenous tissue, in situ hybridization was used to localize MT-I and MT-III mRNA in control and treated mice. MT-I mRNA signal, which was most abundant in the glial cells of the Purkinje cell layer of the cerebellum in control mice, appeared to be enhanced in mice given the MT-I inducers (LPS, Zn, Cd, Dex, ethanol, and KA). MT-I mRNA hybridization signal was also enhanced in the olfactory bulbs from LPS- and Cd-treated mice, while this signal was present but weak in control brains. MT-III mRNA hybridization signals were localized in hippocampus and co-localized with MT-I message in the glial cells of the Purkinje cell layer of the cerebellum. In addition, diffuse MT-III mRNA signals were visible in areas of the cerebral cortex, and in the molecular layer of the cerebellum. Signals for MT-III in hippocampus appeared to be reduced by KA, Dex and LPS treatment, while in the cortical region, MT-III mRNA signals appeared to be enhanced by KA, Cd, and ethanol treatment. In conclusion, both MT-I and MT-III expression in brain appears to be modulated by exogenous treatment, however, the changes are small in relation to those observed in liver. Chemical-induced alterations of MT mRNA are non-uniform throughout the brain, and thus best studied in a region-specific manner.

Altered expression of pro-inflammatory and developmental genes in the fetal brain in a mouse model of maternal infection

Human studies of unexplained cerebral palsy (CP) suggest an association with maternal infection. We used an established model of maternal infection, lipopolysaccharide (LPS) administration, to investigate the molecular changes in the fetal brain that may link maternal infection and CP. We compared gene expression in brains from mouse pups exposed to LPS in utero to those from saline-treated controls. Dams were injected with 50 microg LPS or saline on E18 with surgical delivery from 0.5 to 6h later. Differential gene expression was analyzed in the whole mouse brain using RT-PCR. When compared to control mice, pups exposed to LPS showed increased expression of pro-inflammatory genes monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and interleukin-1beta (IL-1beta), as well as VEGF, a regulator of vascular development and permeability, the anti-apoptotic protein Y-box-binding protein-1 (YB-1), and the neuronal differentiation factor necdin. LPS-exposed mice also showed downregulation of semaphorin 5b and groucho, involved in axon guidance and neurogenesis, respectively, providing evidence that LPS may disrupt normal developmental pathways. These data suggest possible mechanisms for adverse neurological outcomes following maternal infection involving elevated cytokine levels and altered expression of developmental genes in the fetal brain.