Christine Hunter

Effects of anti-VEGF antibody on blood-brain barrier disruption in focal cerebral ischemia.

Since cerebral ischemia increases expression of vascular endothelial growth factor (VEGF) and exogenous VEGF can aggravate BBB disruption in cerebral ischemia, we hypothesized that inhibition of endogenous VEGF would attenuate BBB disruption. To test this hypothesis, rats were mechanically ventilated with isoflurane and a craniotomy (5 mm in diameter) was performed to expose the cerebral cortex. Anti-VEGF antibody was applied topically (75 mug) 1 h before middle cerebral artery (MCA) occlusion and additional anti-VEGF antibody was applied (25 mug) immediately after MCA occlusion (anti-VEGF group). For the control animals, normal saline was applied instead of anti-VEGF antibody on the surface of the cortex (control group). One hour after MCA occlusion, the transfer coefficient (K(i)) of (14)C-alpha-aminoisobutyric acid and volume of (3)H-dextran (70,000 Da) distribution were determined to measure the degree of BBB disruption. There was no significant difference in vital signs, blood gases, and pericranial temperature between the control and the anti-VEGF group. In both of the groups, the K(i) of the ischemic cortex (IC) was higher than that of the corresponding contralateral cortex (CC) (p<0.05). The K(i) of the IC of the anti-VEGF group was significantly lower than that of the IC of the control group (-34%, p<0.05). The K(i) of the CC and pons were similar between these two groups. The data of volume of dextran distribution followed the same pattern as that of K(i) but without a statistical significance. Our data demonstrated that inhibition of endogenous VEGF by topical application of anti-VEGF antibody in the ischemic cortex decreased the K(i) of (14)C-AIB and suggest that endogenous VEGF is in part responsible for the BBB disruption during the early stage of focal cerebral ischemia.

Effects of VEGF and nitric oxide synthase inhibition on blood–brain barrier disruption in the ischemic and non-ischemic cerebral cortex

Abstract Objectives: This study was performed to compare the effects of exogenous vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS) inhibition on blood–brain barrier (BBB) disruption in the ischemic cortex (IC) and non-ischemic contralateral cortex (CC) during the early stage of focal cerebral ischemia in rats. Methods: A middle cerebral artery (MCA) was occluded after a craniotomy in each rat under isoflurane anesthesia. Two more craniotomies were performed over the contralateral non-ischemic hemisphere to expose cerebral cortex. For the control rats, the normal saline patches were applied to all three craniotomy holes (control group). To inhibit NOS, NG-nitro-L-arginine-methyl ester (L-NAME) (10 mg/kg) was administered i.v. 20 minutes after MCA occlusion (L-NAME group). In another group, VEGF (10−10 M) was topically applied 30 minutes after MCA occlusion on the IC as well as one of the holes of the contralateral cortex (VEGF group). To investigate the effects of the combination of VEGF and L-NAME, both L-NAME and VEGF were administered as described above (L-NAME+ VEGF group). The transfer coefficient (Ki) of 14C-α-aminoisobutyric acid and the volume of 3H-dextran (70000 Da) distribution were determined to measure the degree of BBB disruption at 1 hour after MCA occlusion. Results: In the control group, Ki of the IC was significantly higher than the contralateral cortex (CC) (p<0.005). VEGF application increased the Ki of the IC further when compared with the control group (+51%, p<0.05%). L-NAME administration produced no significant decrease in the Ki of the IC when compared with the control group. With L-NAME+ VEGF administration, the Ki of the IC became significantly lower than that of the VEGF alone (−38%, p<0.005). Thus, L-NAME produced a much greater decrease in the Ki of the IC in the VEGF treated than the control animals (p<0.05). In the non-IC, VEGF, L-NAME, or their combination did not affect BBB disruption. The volume of dextran distribution followed a similar pattern to Ki. Discussion: Our data suggest that even in the early stage of focal cerebral ischemia, the degree of BBB disruption in response to the exogenous VEGF is much greater in the ischemic than in the non-IC and that the mechanism of the increase of BBB disruption by VEGF in the IC involves the NOS pathway.

The effects of isoflurane pretreatment on cerebral blood flow, capillary permeability, and oxygen consumption in focal cerebral ischemia in rats.

BACKGROUND: We performed experiments to test whether isoflurane pretreatment produces vascular effects, especially at the levels of arterioles and capillaries affecting regional cerebral blood flow (rCBF), O2 supply and consumption, or capillary permeability in focal cerebral ischemia. Because inducible nitric oxide synthase (iNOS) was implicated as one of the mechanisms of isoflurane preconditioning, the effect of iNOS inhibition on rCBF was also studied. METHODS: Twenty-four hours before middle cerebral artery (MCA) occlusion, rats were pretreated with 2% isoflurane for 30 minutes using an endotracheal tube and mechanical ventilation for the isoflurane preconditioned (IsoPC) group. For the group of iNOS inhibition, aminoguanidine 200 mg/kg was injected IP 30 minutes before isoflurane pretreatment. One hour after MCA occlusion, rCBF was measured using 14C-iodoantipyrine autoradiography. Alternate slices of the tissue were used to determine arteriolar and venular O2 saturation using cryo microspectrophotometry. Capillary permeability was determined by measuring the transfer coefficient (Ki) of 14C-&agr;-aminoisobutyric acid. Additional measurements of rCBF were performed at 3 hours after MCA occlusion. RESULTS: MCA occlusion decreased rCBF and O2 consumption and increased Ki in both the control and the IsoPC groups at 1 hour after MCA occlusion. In the ischemic cortex (IC), the rCBF and O2 consumption were significantly greater in the IsoPC group than in the control group (+40% and +41%, respectively), but they were similar in the contralateral cortex between the 2 groups. There was no difference in Ki between the groups in the IC or in the contralateral cortex. The increase of rCBF in the IC (+50%) was sustained in the IsoPC group at 3 hours after MCA occlusion. With iNOS inhibition, the increase of rCBF in the IC with isoflurane pretreatment became insignificant. CONCLUSIONS: Our data demonstrate that isoflurane pretreatment improved rCBF and increased the regional O2 supply and consumption in the focal ischemic area but did not affect capillary permeability during the early stage of focal cerebral ischemia. The isoflurane-induced increase in rCBF in the ischemic area became insignificant with inhibition of iNOS.

Effects of Erythropoietin on Blood-Brain Barrier Disruption in Focal Cerebral Ischemia

This study was performed to test whether systemically administered erythropoietin (EPO) could attenuate the blood-brain barrier (BBB) disruption in focal ischemia. Rats were injected intraperitoneally with 2,500 IU/kg of recombinant human EPO or normal saline 24 h before middle cerebral artery (MCA) occlusion. The transfer coefficient (Ki) of 14C-α-aminoisobutyric acid was determined to measure the degree of BBB disruption 1 h after MCA occlusion. In the control animals, the Ki of the ischemic cortex (IC) was significantly higher than that of the contralateral cortex (CC; +128%, p = 0.0002). In the EPO-treated animals, the Ki of the IC was not significantly different from that of the CC and was significantly lower (–44%, p = 0.003) than that of the control animals. Our data suggest that MCA occlusion increased BBB disruption, and the disruption was attenuated with EPO pretreatment.

The effects of dexmedetomidine on regional cerebral blood flow and oxygen consumption during severe hemorrhagic hypotension in rats.

BACKGROUND: We performed this study to determine how dexmedetomidine would affect regional cerebral blood flow (rCBF) and microregional O2 consumption during nonhemorrhagic normovolemia and during severe hemorrhagic hypotension in rats. METHODS: Forty-eight male rats were anesthetized with isoflurane and their lungs were mechanically ventilated. Half of the rats were bled to reach a mean arterial blood pressure of 40 to 45 mm Hg and were maintained at this level for at least 30 minutes before rCBF or microregional arterial oxygen saturation (SaO2) and venous oxygen saturation (SvO2) were determined. The other half were not bled and served as nonhemorrhagic controls. Half of each group was given dexmedetomidine 1 &mgr;g/kg/min IV for 45 minutes and the other half was given the same amount of normal saline infusion. The infusion started 10 minutes before blood withdrawal for the hemorrhagic groups. The rCBF was determined using 14C-iodoantipyrine, and the microregional SaO2 and SvO2 were determined using cryomicrospectrophotometry at 45 minutes of infusion. RESULTS: Dexmedetomidine decreased heart rate by 25%, but the decrease of mean arterial blood pressure was not significant. The total amount of blood withdrawn and hemoglobin were similar between the normal saline-treated and the dexmedetomidine-treated groups. In normovolemia, dexmedetomidine significantly decreased rCBF (−58%) in the lateral cortex with a similar percentage decrease (−57%) of calculated O2 consumption. Microregional SvO2 was similar between the normal saline-treated group (62.8% ± 1.3% [mean ± SD]) and the dexmedetomidine-treated group (60.7% ± 1.8%) despite a large difference in rCBF. Hemorrhage significantly decreased rCBF (−44%) in the lateral cortex in the normal saline-treated rats with no significant change in regional cerebrovascular resistance. In contrast, in the lateral cortex of the dexmedetomidine-treated rats, the decrease of rCBF was not significant but there was a significant decrease in regional cerebrovascular resistance. A decrease (−25%) in the O2 consumption was observed in the lateral cortex of the normal saline-treated rats with hemorrhage, but hemorrhage did not decrease O2 consumption in the dexmedetomidine-treated rats. Despite significantly lower rCBF (−34%) in the dexmedetomidine-treated rats, the SvO2 was similar between the normal saline-treated (42.8% ± 2.5%) and the dexmedetomidine-treated rats (43.2% ± 2.7%). CONCLUSIONS: Our data showed that in normovolemia, dexmedetomidine produced a proportionate decrease of rCBF and O2 consumption. Hemorrhage decreased rCBF more than O2 consumption. Dexmedetomidine prevented rCBF and O2 consumption from decreasing after hemorrhage. Our data suggest that dexmedetomidine may help provide optimal O2 supply and consumption balance during hemorrhage.

Effects of deferoxamine on blood–brain barrier disruption and VEGF in focal cerebral ischemia

Abstract Objective: Deferoxamine, an iron chelator, is reported to induce hypoxia-inducible factor 1 (HIF-1) that leads to transcriptional activation of numerous genes including vascular endothelial growth factor (VEGF) that is known to increase blood–brain barrier (BBB) permeability. This study was performed to test whether deferoxamine would disrupt BBB further in focal cerebral ischemia by altering the level of VEGF. Methods: Rats were injected intraperitoneally with normal saline (control group), 300 mg/kg deferoxamine mesylate 18 (deferoxamine 18 group) or 48 (deferoxamine 48 group) hours before middle cerebral artery (MCA) occlusion. The transfer coefficient (Ki) of 14C-α-aminoisobutyric acid (14C-AIB) and the volume of 3H-dextran distribution were determined to measure the degree of BBB disruption 1 hour after MCA occlusion. Immunohistochemistry using a monoclonal VEGF antibody was performed to determine the protein level of VEGF. Results: In all groups of animals, the Ki of the ischemic cortex (IC) was higher than that of the corresponding contralateral cortex (CC). In the deferoxamine 18 group, the Ki of the IC was significantly higher than that in the control group (+52%, p<0.05) or deferoxamine 48 group (+72%, p<0.05). The Ki of the CC of all experimental groups were similar. The volume of dextran distribution of the IC was significantly higher than that of the CC only in the deferoxamine 18 group. The number of areas that were stained with VEGF antibody in the deferoxamine 18 group (106 ± 5/mm2) was significantly higher than that in the control group (54 ± 2/mm2) or deferoxamine 48 group (58 ± 1/mm2). Discussion: Our data suggest that deferoxamine induced an increase in VEGF but that its effect depends on the time of administration. The increase in VEGF by deferoxamine could aggravate the disruption of BBB in focal cerebral ischemia.

Effects of GABAA receptor blockade on regional cerebral blood flow and blood-brain barrier disruption in focal cerebral ischemia

In cerebral ischemia, transmission by the inhibitory neurotransmitter, γ-aminobutyric acid (GABA) is altered. This study was performed to determine whether blockade of GABA(A) receptor would affect regional cerebral blood flow (rCBF) and blood-brain barrier (BBB) permeability in a focal ischemic area of the brain. Rats were anesthetized with isoflurane and mechanically ventilated. Fifteen minutes after a permanent middle cerebral artery (MCA) occlusion, one half of the rats were infused with bicuculline 1mg/kg/min iv for 2 min followed by 0.1mg/kg/min iv to the end of the experiment. The other half were infused with normal saline. At one hour after MCA occlusion, rCBF was determined using ¹⁴C-iodoantipyrine and BBB permeability was determined by measuring the transfer coefficient (Ki) of ¹⁴C-α-aminoisobutyric acid. With MCA occlusion, rCBF was decreased in the ischemic cortex (IC) (-70%) in the control rats. In the bicuculline treated rats, the rCBF of the IC was lower (-48%) than the contralateral cortex but higher than the rCBF of the IC of the control rats (+55%). MCA occlusion increased Ki in the IC of the control rats (+72%) and bicuculline administration increased Ki further (+53%) in the IC. Blockade of GABA(A) receptors did not significantly affect rCBF or BBB permeability in the non-ischemic brain regions under isoflurane anesthesia. Our data demonstrated that blockade of GABA(A) receptors increased rCBF and enhanced the BBB disruption in focal cerebral ischemia. Our data suggest that GABA(A) receptors are involved, at least in part, in modulating rCBF and BBB disruption in focal cerebral ischemia.

Effects of Fentanyl Pretreatment on Regional Cerebral Blood Flow in Focal Cerebral Ischemia in Rats

Background:There are reports that opioid preconditioning induces opioid-receptor-dependent neuroprotection against cerebral ischemia. This experiment was performed to test whether pretreatment with fentanyl, a synthetic primary µ-opioid receptor agonist, would affect the regional cerebral blood flow (rCBF) in focal cerebral ischemia in rats. Methods: Twenty-four hours before permanent and unilateral middle cerebral artery (MCA) occlusion, rats were pretreated with normal saline, 200 µg/kg of fentanyl i.p. or 500 µg/kg of fentanyl i.p. The rats were anesthetized with isoflurane and were mechanically ventilated to cannulate the vessels and to occlude MCA. One hour after MCA occlusion, the rCBF was measured using 14C-iodoantipyrine. Results: The cortical rCBF decreased 1 h after MCA occlusion in all the experimental groups. In the ischemic cortex, the rCBF of the rats treated with 500 µg/kg of fentanyl was significantly greater (+80%, p < 0.05) than that of the control animals. The rCBF of the ischemic cortex of the rats treated with fentanyl 200 µg/kg seemed higher than in the control animals, but the difference was not statistically significant. The rCBF was similar in the nonischemic brain regions such as the contralateral cortex or pons among the experimental groups. Conclusion: Our data demonstrated that pretreatment with fentanyl improved the rCBF in the focal ischemic area without change in rCBF in the nonischemic cortex. Our data suggest that fentanyl could be effective in improving the rCBF in the focal ischemic area when used as a preconditioning agent and that improvement of rCBF could be one of the contributing factors of neuroprotection by opioid preconditioning.

Effects of VEGF on the Blood-Brain Barrier Disruption Caused by Hyperosmolarity

This study was performed to test whether disruption of the blood-brain barrier (BBB) caused by hyperosmolarity could be related to vascular endothelial growth factor (VEGF), using anti-VEGF antibody and ciclopirox olamine (CPX), an inducer of VEGF. CPX 50 mg/kg or normal saline was given intraperitoneally to male Wistar rats 18 h before BBB disruption. Two craniotomies were made on the ipsilateral cortex (IC-1 and IC-2) where the BBB would be disrupted, and a third hole was made on the contralateral cortex (CC) to expose the cortices. We applied normal saline (to IC-1 and the CC) or anti-VEGF antibody (to IC-2) for 90 min before BBB disruption with intracarotid injection of 25% mannitol. The degree of BBB disruption was determined by measuring the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid and the volume of 3H-dextran distribution. The protein levels of VEGF were determined with Western blot analysis. In the control animals, hyperosmolar mannitol significantly increased (415%) the Ki in IC-1. The Ki was attenuated with anti-VEGF antibody application (–28%, p < 0.05). Even though the protein levels of VEGF were strongly increased with CPX pretreatment, this upregulation did not alter the hyperosmolar BBB disruption in the saline- or in the antibody-treated cortex. The data on the volume of dextran distribution followed the same pattern as that of the Ki but without a statistically significant difference between IC-1 and IC-2 in either group. Our data demonstrated that hyperosmolar BBB disruption could be attenuated with anti-VEGF antibody. However, upregulation of VEGF with CPX did not alter the degree of hyperosmolar BBB disruption with or without anti-VEGF antibody treatment. This study suggests that the contribution of VEGF in hyperosmolar BBB disruption is limited.

A new use for the GlideScope.

To the Editor: We report a case in which we used the GlideScope to successfully place a nasogastric tube after all other placement methods had failed. Our 63-yr-old male patient had undergone a right hemicolectomy 2 wk before admission. He presented to the emergency room with a 2-day history of distended abdomen and absence of defecation. The patient was scheduled for an emergent exploratory laparotomy. On arrival in the operating room, he complained of intense abdominal pain and discomfort secondary to abdominal distention. The patient’s airway was classified as Mallampati I, and he was edentulous. We quickly and easily induced anesthesia and intubated the trachea in rapid sequence. We tried and failed several times to place a nasogastric tube, including digitally manipulating its tip and visualizing the posterior oropharynx with Macintosh and Miller blades. Once the GlideScope was brought to the operating room, we easily placed the short GlideScope blade in the pharynx and obtained a clear view of the laryngeal structures and the entrance to the esophagus. We lifted the epiglottis with the GlideScope’s tip and advanced the nasogastric tube manually into the esophagus. The position was verified by surgical palpation in the stomach and return of gastric fluid when aspirated. The GlideScope is designed to assist with challenging intubations by providing a video view of the larynx during intubation. One can master using the GlideScope with a few attempts. The GlideScope adds to our available techniques for passing intransigent orogastric tubes.