Neocortical Spreading Depression Research Articles

Insulin-like growth factor-1 inhibits spreading depression-induced trigeminal calcitonin gene related peptide, oxidative stress & neuronal activation in rat

Migraineurs can show brain hyperexcitability and oxidative stress that may promote headache. Since hyperexcitability can enhance oxidative stress which promotes hyperexcitability, ending this feed-back loop may reduce migraine. Neocortical spreading depression, an animal model of migraine begins with hyperexcitability and triggers oxidative stress in the neocortical area involved and in the trigeminal system, which is important to pain pathway nociceptive activation in migraine. Additionally, oxidative stress causes increased trigeminal ganglion calcitonin gene-related peptide release and oxidative stress can reduce spreading depression threshold. Insulin-like growth factor-1 significantly protects against spreading depression in vitro by reducing oxidative stress and it is effective against spreading depression after intranasal delivery to animals. Here, we used adult male rats and extend this work to study the trigeminal system where insulin-like growth factor-1 receptors are highly expressed. Recurrent neocortical spreading depression significantly increased surrogate markers of trigeminal activation - immunostaining for trigeminal ganglion oxidative stress, calcitonin gene related peptide levels and c-fos in the trigeminocervical complex versus sham. These effects were significantly reduced by intranasal delivery of insulin-like growth factor-1 a day before recurrent neocortical spreading depression. Furthermore, intranasal treatment with insulin-like growth factor-1 significantly reduced naïve levels of trigeminal ganglion calcitonin gene related peptide versus sham with no impact on blood glucose levels. Intranasal delivery of insulin-like growth factor-1 not only mitigates neocortical spreading depression, a cause of migraine hyperexcitability modeled in animals, but also when neocortical spreading depression is triggered by supra-threshold stimuli, insulin-like growth factor-1 effectively reduces nociceptive activation in the trigeminal system.

Intranasally administered IGF-1 inhibits spreading depression in vivo

Spreading depression (SD) is a wave of cellular depolarization that travels slowly through susceptible gray matter brain areas. SD is the most likely cause of migraine aura and perhaps migraine pain, and is a well-accepted animal model of migraine. Identification of therapeutics that can prevent SD may have clinical relevance toward migraine treatment. Here we show that insulin-like growth factor-1 (IGF-1) significantly inhibited neocortical SD in vivo after intranasal delivery to rats. A single dose of IGF-1 inhibited SD within an hour, and continued to protect for at least seven days thereafter. A two-week course of IGF-1, administered every third day, further decreased SD susceptibility and showed no aberrant effects on glial activation, nasal mucosa, or serum markers of toxicity. SD begets SD in vitro by mechanisms that involve microglial activation. We add to this relationship by showing that recurrent SD in vivo increased susceptibility to subsequent SD, and that intervention with IGF-1 significantly interrupted this pathology. These findings support nasal administration of IGF-1 as a novel intervention capable of mitigating SD susceptibility, and as a result, potentially migraine.

Changes in Magnetization Transfer MRI Correlate with Spreading Depression–Induced Astroglial Reactivity and Increased Protein Expression in Mice

Gliosis refers to a range of glial cell transformations that vary according to specific brain pathologic states. Disease, however, is not a prerequisite for gliosis because glial reactivity may also be seen in regions of increased physiologic activity. Our study tests the hypothesis that high-field-strength magnetization transfer MRI is a sensitive technique for detecting transient glial reactivity after experimental spreading depression, a relatively benign perturbation unaccompanied by cell injury. Unilateral neocortical spreading depression was elicited in mouse cerebral hemispheres and confirmed by transcranial blood flow and extracellular potential measurements. After 3 days, mice were imaged at 4 T using magnetization transfer techniques. Astroglial reactivity was determined immunohistochemically, and protein expression in control and experimental hemispheres was compared using proteomic techniques. Sixteen ([mean +/- SD] +/- 3) spreading depressions (n = 10) were recorded in experimental hemispheres. Spreading depression was never observed in control hemispheres. At 3 days, an 8% decrease (p < 0.05, n = 4) in magnetization transfer signal intensity was measured in experimental hemispheres, which was associated with a 37% increase (p < 0.001, n = 4) in the intensity of glial fibrillary acidic protein staining. Proteomic analysis performed 3 days after the induction of spreading depression showed upregulation of at least 56 proteins, including extracellular and intracellular elements. Magnetization transfer at 4.0-T MRI is a sensitive method for detecting glial reactivity and changes in protein expression not associated with cell injury. These results suggest magnetization transfer MRI techniques may have potential for detecting glial reactivity in physiologic processes such as learning and in early disease states.

Possible mechanism of c-fos expression in trigeminal nucleus caudalis following cortical spreading depression

Cortical spreading depression (CSD) is characterized by a transient, reversible depression of EEG activity which advances across the cortical surface at a velocity of 2–5 mm/min. CSD was originally linked to the aura phase of migraine, but recently also to migraine headache. The theory is that CSD activates meningeal trigeminal C-fibers causing neurogenic inflammation and pain (Moskowitz, M.A., Nozaki, K. and Kraig, R.P., Neocortical spreading depression provokes the expression of c-fos protein-like immunoreactivity within trigeminal nucleus caudalis via trigeminovascular mechanisms, J. Neurosci., 13 (1993) 1167–1177). The present study is an examination of the proposed link between CSD elicited in rats and activation of trigeminal nerve fibers. Multiple CSDs were elicited unilaterally for 1 h by KCl injections (1 M, 5 μl) into the right hemisphere, while NaCl (1 M, 5 μl) was injected into the left as control. After an additional 1 h the animals were sacrificed and trigeminal activation assessed by the expression of c-fos in trigeminal nucleus caudalis (TNC) using immunohistochemistry. The correlation between the number of CSDs and the extent of c-fos expression was determined. In addition the effect of sumatriptan (0.3 mg/kg) and morphine (3 mg/kg) given i.v. 30 min before elicitation of CSD was evaluated. CSD caused increased c-fos expression in lamina I and II of TNC where C-fibers end, the response being greater ipsilaterally. Morphine, but not sumatriptan, reduced c-fos expression in both the ipsilateral and contralateral TNC by 71% ( P<0.05 and P=0.19, respectively), confirming that nociceptors have been activated. No positive correlation was seen between the number of CSDs and the extent of c-fos expression in TNC. Instead we observed a positive, linear correlation between the number of KCl injections and the extent of c-fos expression in TNC (correlation coefficient r=0.709, P<0.05). We suggest that the C-fiber activation observed is caused by hyperosmolar KCl/NaCl and not CSD. Hence, our results do not support the hypothesis of Moskowitz et al. (Moskowitz, M.A., Nozaki, K. and Kraig, R.P., Neocortical spreading depression provokes the expression of c-fos protein-like immunoreactivity within trigeminal nucleus caudalis via trigeminovascular mechanisms, J. Neurosci., 13 (1993) 1167–1177) which links CSD with migraine headache.

Long-term elevation of cyclooxygenase-2, but not lipoxygenase, in regions synaptically distant from spreading depression.

Eicosanoids, produced from arachidonic acid by cyclooxygenases (COXs) and lipoxygenases (LIPOXs), are involved in numerous brain processes. To explore if brief and noninjurious stimuli chronically alter expression of these enzymes, we examined the induction of COX-2 and LIPOX expression following unilateral neocortical spreading depression (SD). Expression was examined over time and in regions not experiencing SD (hippocampus) but synaptically connected to the site of stimulation (cortex). One hundred six male Wistar rats had SD induced via microinjection of 0.5 M KCl (0.5 M NaCl for sham) into left parietal cortex every 9 minutes for 1 or 3 hours. One hour before SD some animals received dexamethasone (Dex), mepacrine (Mep), indomethacin (Indo), nordihydroguaiaretic acid (Ndga), phenylephrine (Pe), sodium nitroprusside (Snp) with Pe, or N omega-nitro-L-arginine methyl ester (Lnam). Animals survived for 0, 3, or 6 hours, or 1, 2, 3, 7, 14, 21, or 28 days. Brains were processed immunohistochemically for COX-2 and LIPOX, and the optical density (OD) of the left and right cortex, dentate gyrus (DG), CA3, and CA1 immunoreactivity (IR) were measured. Induction was expressed as the log of left divided by right side OD for each region. COX-2 IR in the left cortex was elevated rapidly and was sustained for 21 days following SD. COX-2 IR was also elevated in the ipsilateral hippocampus not experiencing SD, with the rank order of induction as follows: DG > CA3 > CA1. Dex, Snp, and/or Pe significantly reduced the induction of COX-2. No changes in LIPOX IR were observed. These results show that long-term changes in COX-2 expression are induced by SD and these changes decrease with synaptic distance. Benign stimuli increase COX-2 expression and thus may influence brain function for extended periods and at distant locations.

Eicosanoids and nitric oxide influence induction of reactive gliosis from spreading depression in microglia but not astrocytes.

Microglia and astrocytes are transformed into reactive glia (RG) by brain disease and normal function. Eicosanoids and nitric oxide (NO), two intercellular mediators, may influence gliosis. We investigated how drugs that alter production of these paracrine signals effect induction of glial reactivity from spreading depression. Unilateral (left) neocortical spreading depression was induced in 95 halothane anesthetized rats by intracortical injections of 0.5 M KCl, with or without drug treatment (five animals/group). Immunohistochemical staining (IS) intensity using the OX-42 and anti-glial fibrillary acidic protein (GFAP) antibodies determined reactivity in microglia and astrocytes, respectively. After 3 days, brains were processed for OX-42 and GFAP-IS and mean optical densities (OD) of IS were measured. Average OD's (for OX-42) and the log ratio (left/right) of OD's (OX-42 and GFAP) were compared to normal animals. Spreading depression induced significant log ratios for both OX-42- and GFAP-IS (P's < 0.01). However, dexamethasone (a glucocorticoid), nordihydroguaiaretic acid (a lipoxygenase inhibitor), and nitroprusside (a NO donor) prevented significant left sided and log ratio OD values for microglia (P's > 0.05). L-Name, a NO synthase inhibitor, caused significant increases in left and right OD's for microglia (P's < 0.05). Mepacrine, a phospholipase A2 inhibitor, Indomethacin, a cyclooxygenase inhibitor, and phenylephrine, an adrenergic agonist, did not prevent induction of significant OX-42 log ratios (P's < 0.01, 0.05, 0.01), and resulted in increases in left side OD's (P's < 0.01, 0.05, 0.05). Significant GFAP log ratios occurred after spreading depression in all drug groups, P's < 0.01. Thus, induction of reactivity in microglia is more sensitive to eicosanoids and NO than in astrocytes.

Neocortical spreading depression provokes the expression of c-fos protein-like immunoreactivity within trigeminal nucleus caudalis via trigeminovascular mechanisms

The effects of neocortical spreading depression (SD) on the expression of immunoreactive c-fos protein were examined within the superficial laminae of trigeminal nucleus caudalis (TNC), a brainstem region processing nociceptive information. KCl was microinjected into the left parietal cortex at 9 min intervals over 1 hr, and SD was detected by a shift in interstitial DC potential within adjacent frontal cortex. The stained cells in lower brainstem and upper cervical spinal cord were counted on both sides after tissues were sectioned (50 microns) and processed for c-fos protein-like immunoreactivity (LI) using a rabbit polyclonal antiserum. C-fos protein-LI was visualized in the ventrolateral TNC, chiefly in laminae I and Ilo and predominantly within spinal segment C1-2 (e.g., -1.5 to -4.5 mm from obex) ipsilaterally. SD significantly increased cell staining within ipsilateral TNC. The ratio of cells in laminae I and Ilo on the left: right sides was 1.32 +/- 0.13 after 1 M KCl, as compared to 1.06 +/- 0.05 in control animals receiving 1 M NaCl instead of KCl microinjections (p < 0.01). The ratio was reduced to an insignificant difference after chronic surgical transection of meningeal afferents and recurrent SD (1.09 +/- 0.11). Pretreatment with intravenous sumatriptan, a 5-HT1-like receptor agonist that selectively blocks meningeal C-fibers and attenuates c-fos protein-LI within TNC after noxious meningeal stimulation, also reduced the ratio to an insignificant difference (1.10 +/- 0.09). Sumatriptan or chronic surgical transection of meningeal afferents, however, did not reduce the ability of KCl microinjections to induce SD. On the other hand, combined hyperoxia and hypercapnia not only reduced the number of evoked SDs from 6.3 +/- 1.0 to 2.5 +/- 1.2 after 0.15 M KCl microinjection, but also significantly (p < 0.01) reduced associated c-fos protein-LI in TNC. These data indicate that multiple neocortical SDs activate cells within TNC. The increase in c-fos protein-LI, observed predominantly ipsilaterally, was probably mediated by SD-induced stimulation of ipsilaterally projecting unmyelinated C-fibers innervating the meninges. If true, this is the first report demonstrating that neurophysiological events within cerebral cortex can activate brainstem regions involved in the processing of nociceptive information via trigeminovascular mechanisms.

Spreading depression increases immunohistochemical staining of glial fibrillary acidic protein

Reactive astrocytosis is a process by which astrocytes respond to brain injury by showing an increase in glial fibrillary acidic protein (GFAP) staining that is associated with hypertrophy and/or hyperplasia of these cells. Because spreading depression (SD) is a perturbation uncomplicated by neuronal necrosis and is seen in both in vivo and in vitro neural structures, we sought to determine whether SD was a sufficient stimulus to induce enhanced GFAP staining. SD was elicited in anesthetized rats by application of KCI to parietal cortex for 3 hr; equimolar NaCI was applied to contralateral cortex. SD was confirmed by monitoring DC potentials in frontal neocortices. Animals were allowed to recover for 48 hr, and their brains were processed for semiquantitative and computer-based analyses of GFAP staining intensity. Experimental GFAP staining was referenced to contralateral control levels. Neocortical SD (13-37 SDs) was associated with a significant (p less than 10(-4)), 43% increase in GFAP staining intensity, which remained statistically greater than normal for more than 2 weeks. If SD was inhibited by combined hyperoxia and hypercarbia, only a nonsignificant (p greater than 0.20), 7% increase in GFAP staining was seen. Thus, SD may be a useful physiologic process with which to begin to explore the cellular mechanisms that induce the transformation of normal astrocytes into reactive species.

Circling and consumatory behavior induced by striatal and neocortical spreading depression

Single waves of spreading depression (SD) were induced in freely moving Sprague Dawley rats by microinjection of KCl solution into either neocortex or caudate nucleus. DC recordings of slow potential changes (SPC) revealed that a wave of SD propagates from the neocortex to the caudate nucleus in only a very few cases (12.5% of trials, involving 4.3% of the SPC waves). This result contrasts with earlier reported higher incidences of neocortical-striatal transmission of SD, and suggests that such transmission may vary across rat strains. Invasion of the caudate nucleus by a wave of neocortical SD was accompanied by contralateral circling in 50% of the cases. Similarly, SD induced by injection of KCl into the striatum was accompanied by circling in 43% of trials. It was also found that neocortical SD-induced feeding generally occurred only upon recovery of neocortex from the slow potential negativity, and that invasion of the caudate nucleus by SD is not involved in the elicited feeding phenomenon.

Effect of hippocampal and neocortical spreading depression on rat shuttle behavior in four different experimental paradigms

As shown in previous papers, rat shuttle responses to a buzzer (SBs) depend on the operation of three main factors: a state of drive (D) determined by the presence of footshocks in the experimental situation; the pairing (i.e., fixed temporal relation) of buzzers and shocks (P); the contingency (C) of shocks upon SBs. Four tests were studied in which these factors operate in various combinations: (a) D test: 50 buzzers and 25 shocks at random intervals and in random sequence; (b) DP test (Pavlovian conditioning): 50 buzzers paired (contiguous) to shocks on trials, regardless of whether SBs are performed or not; (c) DC test: 50 buzzers, each followed by a shock at a randomly variable interval unless there is an SB on that trial; (d) DPC test (two-way avoidance): 50 buzzers paired to shocks only on those trials in which there is no SB. The effect of hippocampal and neocortical spreading depression on these tests was studied. Spreading depression was induced by the application of KCl crystals through implanted guide cannulae, and was ascertained by EEG recording. Application of KCl on the hippocampus caused both hippocampal and neocortical spreading depression; whereas when it was applied on the neocortex there was only spreading depression in this structure. The following behavioral responses were measured: SBs, jumping to the buzzer, and intertial shuttling. Neocortical spreading depression had no effect on any of these parameters. Hippocampal spreading depression caused an increase of SBs and of intertrial crossings in all four tests, and a depression of jumping responses in the DP test. The increase of SBs was more marked in the D and DC tests. In fact, in the animals with hippocampal spreading depression differences in SB performance between DP and D, or between DPC and DC, were very small and not significant; this was not the case in any of the other groups. Since, as shown previously both DP - D and DPC - DC are a measure of the P factor, these data suggest that the hippocampus normally exerts a tonic inhibitory influence on the operation of D, and that at the same time it plays a role in the operation of P. The results are consistent both with hypotheses which attribute to the hippocampusa role in behavioral inhibition, and with those which consider that it processes stimulus-stimulus dependent information.

Nonretention of a visual conditioned heart-rate response under neocortical spreading depression.

Nonretention of a visual conditioned heart-rate response under neocortical spreading depression.

The value of ultramicro-analysis of lactic acid in tracing the penetration of Leão's cortical spreading depression to subcortical areas

Passage of neocortical spreading depression to different cortical and subcortical structures has been studied in rats using the increased lactic acid level as an indicator of the spreading depression. The lactic acid was assessed by the modified Bonting method for determination of lactic acid in submilligram amounts of brain-tissue. Penetration of the spreading depression into the area retrosplenialis agranularis took place in 75% of cases (n = 17), and into the area retrosplenialis granularis, in 47% (n = 17). This segment of entorhinal area which is in contact with amygdalar nuclei was invaded by the spreading depression in 80% of all cases (n = 24) and the distant one in 35% (n = 24). No change in the lactic acid could be demonstrated in the hippocampus (n = 11). The cortical spreading depression penetrates in 100% of cases (n = 6) to the amygdala, and in 55% of cases (n = 11) to the striatum. These results confirm the data obtained with the use of electrophysiological methods.

Electroencephalographic signs of anesthesia and sleep during neocortical spreading depression in rats

1. 1. During bilateral reversible functional elimination of the neocortex evoked by spreading depression (SD) no high voltage slow activity occurs in the mesencephalic tegmentum and dorsal hippocampus of rats with chronically implanted electrodes, after administration of synchronizing drugs (allobarbital, thiopental, chloralose: 40–50 mg/kg; atropine; 6 mg/kg) and during physiological sleep. Unilateral neocortical SD does not interfere with generation of this type of EEG activity. 2. 2. During bilateral neocortical SD we have not seen the electrophysiological manifestations of phase 2 of sleep (paradoxical phase), regular theta activity (4–8 c/sec) in the hippocampus, occurring spontaneously. It is possible to evoke theta activity as a sign of “arousal” by external stimuli or reticular stimulation even in this condition. 3. 3. During unilateral SD the cyclic alternation of both sleep phases (generalized high voltage slow activity — phase 1, and neocortical desynchronization with hippocampal theta activity — phase 2) remains present. Nevertheless, phase 2 becomes significantly shorter and phase 1 significantly longer in duration than in control cycles. The spontaneous arousal reaction following phase 1 becomes significantly longer during unilateral SD. Also the incidence of spontaneous arousal reaction during phase 1 increases in these conditions.

INFLUENCE OF DRUGS AND NEOCORTICAL SPREADING DEPRESSION OF HIPPOCAMPAL 'AROUSAL REACTION'.

In leichter Barbiturat- oder Chloralosenarkose wird dieϑ-Aktivitat im Hippocampus, durch aussere oder retikulare Stimulierung induziert, von einer desynchronisierten Hippocampus-Aktivitat abgelost. Die Amplitude dieser durch Anasthetika hervorgerufenen langsamen hohen Wellen wird wahrend der neocortikal sich ausbreitenden Depression reversibel verkleinert.