Suyue Pan

A Modified Technique for Culturing Primary Fetal Rat Cortical Neurons

The study explored a modified primary culture system for fetal rat cortical neurons. Day E18 embryos from pregnant Sprague Dawley rats were microdissected under a stereoscope. To minimize enzymatic damage to the cultured neurons, we applied a sequential digestion protocol using papain and Dnase I. The resulting sifted cell suspension was seeded at a density of 50,000 cells per cm2 onto 0.1 mg/mL L-PLL-covered vessels. After a four-hour incubation in high-glucose Dulbeccos Modified Eagles Medium (HG-DMEM) to allow the neurons to adhere, the media was changed to neurobasal medium that was refreshed by changing half of the volume after three days followed by a complete medium change every week. The cells displayed progressively robust neurite extension, and nonneuronal-like cells could barely be detected by five days in vitro (DIV); cell growth was still substantial at 14 DIV. Neurons were identified by β-tubulin III immunofluorescence, and neuronal purity within the cultures was assessed at over 95% by both flow cytometry and by dark-field counting of β-tubulin III-positive cells. These results suggest that the protocol was successful and that the high purity of neurons in this system could be used as the basis for generating various cell models of neurological disease.

Combination of mild hypothermia with neuroprotectants has greater neuroprotective effects during oxygen-glucose deprivation and reoxygenation-mediated neuronal injury

Co-treatment of neuroprotective reagents may improve the therapeutic efficacy of hypothermia in protecting neurons during ischemic stroke. This study aimed to find promising drugs that enhance the neuroprotective effect of mild hypothermia (MH). 26 candidate drugs were selected based on different targets. Primary cultured cortical neurons were exposed to oxygen-glucose deprivation and reoxygenation (OGD/R) to induce neuronal damage, followed by either single treatment (a drug or MH) or a combination of a drug and MH. Results showed that, compared with single treatment, combination of MH with brain derived neurotrophic factor, glibenclamide, dizocilpine, human urinary kallidinogenase or neuroglobin displayed higher proportion of neuronal cell viability. The latter three drugs also caused less apoptosis rate in combined treatment. Furthermore, co-treatment of those three drugs and MH decreased the level of reactive oxygen species (ROS) and intracellular calcium accumulation, as well as stabilized mitochondrial membrane potential (MMP), indicating the combined neuroprotective effects are probably via inhibiting mitochondrial apoptosis pathway. Taken together, the study suggests that combined treatment with hypothermia and certain neuroprotective reagents provide a better protection against OGD/R-induced neuronal injury.

Interrupted intracarotid artery cold saline infusion as an alternative method for neuroprotection after ischemic stroke

OBJECT Intracarotid artery cold saline infusion (ICSI) is an effective method for protecting brain tissue, but its use is limited because of undesirable secondary effects, such as severe decreases in hematocrit levels, as well as its relatively brief duration. In this study, the authors describe and investigate the effects of a novel ICSI pattern (interrupted ICSI) relative to the traditional method (uninterrupted ICSI). METHODS Ischemic strokes were induced in 85 male Sprague-Dawley rats by occluding the middle cerebral artery for 3 hours using an intraluminal filament. Uninterrupted infusion groups received an infusion at 15 ml/hour for 30 minutes continuously. The same infusion speed was used in the interrupted infusion groups, but the whole duration was divided into trisections, and there was a 20-minute interval without infusion between sections. Forty-eight hours after reperfusion, H & E and silver nitrate staining were utilized for morphological assessment. Infarct sizes and brain water contents were determined using H & E staining and the dry-wet weight method, respectively. Levels of neuron-specific enolase (NSE), S100β protein, and matrix metalloproteinase 9 (MMP-9) in the serum were determined using enzyme-linked immunosorbent assay. Neurological deficits were also evaluated. RESULTS Histology showed that interrupted ICSI did not affect neurons or fibers in rat brains, which suggests that this method is safe for brain tissues with ischemia. The duration of hypothermia induced by interrupted ICSI was longer than that induced via the traditional method, and the decrease in hematocrit levels was less pronounced. There were no differences in infarct size or brain water content between uninterrupted and interrupted ICSI groups, but neuron-specific enolase and matrix metalloproteinase 9 serum levels were more reduced after interrupted ICSI than after the traditional method. CONCLUSIONS Interrupted ICSI is a safe method. Compared with traditional ICSI, the interrupted method has a longer duration of hypothermia and less effect on hematocrit and offers more potentially improved neuroprotection, thereby making it more attractive as an infusion technique in the clinic.

Therapeutic time window of hypothermia is broader than cerebral artery flushing in carotid saline infusion after transient focal ischemic stroke in rats

Abstract Object: Intracarotid cold saline infusion (ICSI) protects against ischemic stroke not only due to the resulting hypothermia, but also as a result of the cerebral artery flushing. To assess the relative benefit of hypothermia and cerebral artery flushing in neuroprotection, hypothermic and normothermic saline infusions were administrated over a serial time points after the initiation of reperfusion in a rat ischemia model. Methods: Ischemic strokes were induced in Sprague–Dawley rats (n = 115) by occluding the middle cerebral artery for 2 hours using an intraluminal filament. In the hypothermic groups, the brain temperature was lowered to 33–34°C for 20 minutes by ICSI at three time points (0, 1, and 2 hours) after reperfusion. Correspondingly, in the normothermic groups, the brain temperature was maintained at normal levels during intracarotid normothermic saline infusion (INSI) for 20 minutes at the same time points. After 48-hour reperfusion, infarct sizes and brain water contents were determined using 2,3,5-triphenyltetrazolium chloride (TTC) staining and the dry–wet weight method, respectively. Levels of neuron-specific enolase (NSE), S100beta, and matrix metalloproteinase 9 (MMP9) in the serum were measured by enzyme-linked immunoassay (ELISA). Neurological deficits were also evaluated. Results: Immediate infusion after the onset of reperfusion (0 hour) did not result in significant difference for reductions of infarct sizes, neurological deficits or S100beta serum levels between ICSI and INSI groups, compared with the non-infusion group. However, brain water content and NSE serum level were significantly lower in the ICSI group than the non-infusion group. When the infusions were started 1 hour after reperfusion, both ICSI and INSI infusions still reduced the infarct sizes, but only ICSI significantly decreased the brain water content, neurological deficits and S100beta serum level. All therapeutic effects of INSI disappeared when infusions were started 2 hours after reperfusion, whereas infarct size, neurological deficits and S100beta serum level were still reduced significantly in ICSI group, compared with the non-infusion group. Conclusions: The neuroprotection of hypothermia and cerebral artery flushing induced by selective carotid infusion after ischemia weakens as the length of time between the reperfusion and infusion increases. The therapeutic time window of brain hypothermia induced by cold saline infusion is broader than cerebral artery flushing induced by normothermic saline infusion.

Glibenclamide Improves Survival and Neurologic Outcome After Cardiac Arrest in Rats.

Objectives:Glibenclamide confers neuroprotection in animal models as well as in retrospective clinical studies. This study determines whether glibenclamide improves outcome after cardiac arrest in rats. Design:Prospective randomized laboratory study. Setting:University research laboratory. Subjects:Male Sprague-Dawley rats (n = 126). Interventions:Rats successfully resuscitated from 8-minute asphyxial cardiac arrest were randomized to glibenclamide or vehicle group. Rats in the glibenclamide group were intraperitoneally administered glibenclamide with a loading dose of 10 &mgr;g/kg at 10 minutes and a maintenance dose of 1.2 &mgr;g at 6, 12, 18, and 24 hours after return of spontaneous circulation, whereas rats in the vehicle group received equivalent volume of vehicle solution. Measurements and Main Results:Survival was recorded every day, and neurologic deficit scores were assessed at 24, 48, and 72 hours and 7 days after return of spontaneous circulation (n = 22 in each group). Results showed that glibenclamide treatment increased 7-day survival rate, reduced neurologic deficit scores, and prevented neuronal loss in the hippocampal cornu ammonis 1 region. To investigate the neuroprotective effects of glibenclamide in acute phase, we observed neuronal injury at 24 hours after return of spontaneous circulation and found that glibenclamide significantly decreased the rate of neuronal necrosis and apoptosis. In addition, glibenclamide reduced the messenger RNA expression of tumor necrosis factor-&agr; and monocyte chemoattractant protein-1 in the cortex after return of spontaneous circulation. Furthermore, the sulfonylurea receptor 1 and transient receptor potential M4 heteromers, the putative therapeutic targets of glibenclamide, were up-regulated after cardiac arrest and cardiopulmonary resuscitation, indicating that they might be involved in neuroprotective effect of glibenclamide. Conclusions:Glibenclamide treatment substantially improved survival and neurologic outcome throughout a 7-day period after return of spontaneous circulation. The salutary effects of glibenclamide were associated with suppression of neuronal necrosis and apoptosis, as well as inflammation in the brain.

Serum levels of procalcitonin as a biomarker for differentiating between sepsis and systemic inflammatory response syndrome in the neurological intensive care unit.

We explored the value of procalcitonin (PCT) to differentiate sepsis from systemic inflammatory response syndrome (SIRS), and determine sepsis severity in the neurological intensive care unit (NICU). Blood samples were measured for C-reactive protein (CRP) and PCT levels upon NICU admission, on the day of diagnosis of SIRS or sepsis, and at 3 and 7 days after diagnosis. We found that there were significant differences in serum levels of CRP and PCT as well as Glasgow Coma Scale (GCS) score upon admission between the SIRS and sepsis groups (p<0.05). CRP and white blood cell levels were not significantly different when attempting to differentiate sepsis severity (p>0.05). Multiple comparisons showed that significant differences in serum PCT levels were observed between sepsis and severe sepsis groups, as well as sepsis and septic shock groups (p<0.05). We obtained the highest sensitivity and specificity for SIRS and sepsis with cut-off values of 2 ng/mL for PCT, 44 mg/dL for CRP, and 4 for the GCS. There were no differences in CRP and PCT levels between cerebrovascular disease and non-cerebrovascular disease groups (p>0.05). No differences were found between viral and bacterial meningitis groups (p>0.05). PCT levels are valuable in discriminating sepsis from SIRS and determining sepsis severity in critically ill patients with neurological disease.

Intermittent hypothermia is neuroprotective in an in vitro model of ischemic stroke.

Objective: To investigate whether the intermittent hypothermia (IH) protects neurons against ischemic insult and the potential molecular targets using an in vitro ischemic model of oxygen glucose deprivation (OGD). Methods: Fetal rat cortical neurons isolated from Day E18 rat embryos were subjected to 90-min OGD and hypothermia treatments during reoxygenation before examining the changes in microscopic morphology, cell viability, microtubule- associated protein 2 (MAP-2) release, intracellular pH value and calcium, reactive oxygen species (ROS) generation, mitochondrial membrane potential (△Ψm) and neuronal death using cell counting kit (CCK-8), enzyme-linked immunosorbent assay (ELISA), BCECF AM, Fluo-3 AM, DCFH-DA and dihydroethidium (DHE), JC-1 staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), respectively. Results: 90-min OGD induced morphologic abnormalities, cell viability decline, MAP-2 release, intracellular acidosis, calcium overload, increased ROS generation, △Ψm decrease and cell death in primary neurons, which was partially inhibited by continuous hypothermia (CH) and intermittent hypothermia (IH). Interestingly, 6-h CH was insufficient to reduce intracellular calcium overload and stabilize mitochondrial membrane potential (△Ψm), while 12-h CH was effective in reversing the above changes. All IH treatments (6×1 h, 4×1.5 h or 3×2 h) effectively attenuated intracellular free calcium overload, inhibited ROS production, stabilized mitochondrial membrane potential (△Ψm) and reduced delayed cell death in OGD-treated cells. However, only IH intervals longer than 1.5 h appeared to be effective in preventing cell viability loss and intracellular pH decline. Conclusion: Both CH and IH were neuroprotective in an in vitro model of ischemic stroke, and in spite of shorter hypothermia duration, IH could provide a comparable neuroprotection to CH.

Glibenclamide Is Comparable to Target Temperature Management in Improving Survival and Neurological Outcome After Asphyxial Cardiac Arrest in Rats

Background We previously have shown that glibenclamide (GBC), a sulfonylurea receptor 1–transient receptor potential M4 (SUR1‐TRPM4) channel inhibitor, improves survival and neurological outcome after asphyxial cardiac arrest and cardiopulmonary resuscitation (ACA/CPR). Here, we further compare the efficacy of GBC with target temperature management (TTM) and determine whether the efficacy of GBC is affected by TTM. Methods and Results Male Sprague‐Dawley rats (n=213) subjected to 10‐minute ACA/CPR were randomized to 4 groups after return of spontaneous circulation (ROSC): normothermia control (NT); GBC; TTM; and TTM+GBC. Survival, neurodeficit scores, histological injury, as well as the expressions of SUR1 and TRPM4 were evaluated. The 7‐day survival rate was 34.4% (11 of 32) in the NT group, 65% (13 of 20) in the GBC group, 50% (10 of 20) in the TTM group, and 70% (14 of 20) in the TTM+GBC group. Rats that received either GBC, TTM alone, or in combination showed less neurological deficit than NT control at 24, 48, and 72 hours and 7 days after ROSC. Moreover, TTM or GBC ameliorated neuronal degeneration and glial activation in the hippocampal CA1 region with similar efficacy, whereas the combination of them had a trend toward better effect. The subunits of SUR1‐TRPM4 heterodimers were both strongly upregulated after ACA/CPR and expressed in multiple types of brain cells, but partly suppressed by TTM. Conclusions GBC is comparable to TTM in improving survival and neurological outcome after ACA/CPR. When GBC is given along with TTM, less histological injury tended to be achieved.

Guillain-Barré syndrome following sepsis after stereotactic aspiration for spontaneous pontine hemorrhage.

A 52-year-old female was treated with CT-guided stereotactic aspiration for acute spontaneous pontine hemorrhage. On postoperative day 7, the patient was complicated by Acinetobacter baumannii sepsis. As sepsis was stabilized, she developed flaccid weakness and autonomic dysfunction on postoperative day 21. Investigations including neurophysiological studies and cerebral spinal fluid analysis prompted the diagnosis of acute motor axonal neuropathy, a variant of Guillain–Barré syndrome. Intravenous administration of immunoglobulin resolved her potentially life-threatening autonomic instability. At 1-year follow-up, she was able to stand with significant assistance. Although Guillain–Barré syndrome rarely occurs, clinicians should be alert to the possibility of this potentially life-threatening consequence after cranial surgery with severe respiratory infection.

S100β as a biomarker for differential diagnosis of intracerebral hemorrhage and ischemic stroke

Objective: To explore the efficacy of S100 calcium-binding protein B (S100β) in differentiating between intracerebral hemorrhage (ICH) and ischemic stroke (IS). Methods: From June 2014 to July 2015, 46 ICH and 71 IS patients who had undergone computed tomography (CT) scans were enrolled. Patients’ neurological deficits were evaluated by the National Institutes of Health stroke scale (NIHSS), and the modified Rankin scale (mRS) was used to assess functional disability 90 days after discharge. Plasma S100β was measured from a blood sample drawn upon arrival at the emergency department. Results: The plasma S100β concentration in the ICH group was significantly higher than in the IS group (p < 0.001). There were only significant correlations between S100β and hemorrhage volume (r = 0.820, p < 0.001), NIHSS score (r = 0.389, p = 0.008), and mRS (r = 0.732, p < 0.001) in the ICH group. Furthermore, receiver-operating characteristic (ROC) curve analysis revealed that an S100β concentration of 67 pg/ml yielded an area under the curve (AUC) of 0.903 with 95.7% sensitivity and 70.4% specificity in differentiating between ICH and IS. In the ICH group, the plasma S100β concentration was significantly elevated in patients with poor functional outcome vs. those with favorable functional outcome (p < 0.001). ROC curve analysis showed that an S100β concentration of 133 pg/ml yielded an AUC of 0.924 with 100% sensitivity and 76.2% specificity in identifying ICH patients with poor functional outcome. Conclusion: S100β could serve as a potential biomarker for differentiating between ICH and IS and predicting short-term functional outcome after ICH.