J Neurosurg 110:94–100, 2009 Hydrogen peroxide–induced stroke: elucidation of the mechanism in vivo Laboratory investigation Melike Mut, M.D., Ph.D.,1 Muge Yemisci, M.D., Ph.D., 2 Yasemin Gursoy-Ozdemir, M.D., Ph.D., 2 and Ugur Ture, M.D. 3 1 Department of Neurosurgery, 2Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara; and 3Department of Neurosurgery, Yeditepe University, Istanbul, Turkey Object. Hydrogen peroxide (H2O2) is used as a hemostatic agent in many neurosurgery centers. The authors used a 3% H2O2 solution for final hemostasis after removal of a left insular tumor. Immediately afterward, air bubbles were observed within the lumen of the polar temporal artery. Postoperative MR imaging revealed punctate areas of infarction in the lenticulostriate artery territory. The authors designed an experimental study to elucidate the mechanism of remote O2 emboli and reactive O2 species–related vasoactive responses and thrombus formation. Methods. In this study, H2O2 irrigation was used in mice with either an intact pial layer or after the pia mater was removed through a corticotomy. Normal saline irrigation was used in the corresponding control groups. Vessels were examined for intravascular O2 emboli under the microscope. Tissue sections were then obtained and stained with H & E and the 3-nitrotyrosine (3-NT) antibody to evaluate intravascular thrombus formation and peroxynitrite reaction, respectively. Results. Multiple bubbles were observed within the lumen of the vessels after exposure to H2O2 regardless of whether the pial layer was destroyed or intact. Immunofluorescent staining for 3-NT showed an abundant positive reaction in the vessel walls of all animals exposed to H2O2 as well as vascular occlusion with acute thrombus formation. Samples taken from the animals that received saline showed no positive staining for 3-NT and no vascular occlusion. Conclusions. Exposure to H2O2 may cause serious ischemic complications. The formation of peroxynitrite may cause vasoactive responses to H2O2 and platelet aggregation/thrombus formation, and the free diffusion of H2O2 through the vessel walls and its conversion to water and O2 leads to O2 bubbles within the closed vessel lumen. If used intradurally, H2O2 may have deleterious ischemic effects, and it can only be used carefully in open extradural spaces. (DOI: 10.3171/2008.3.17434) Key Words • hydrogen peroxide • insula • lenticulostriate artery • mouse • oxygen embolus peroxynitrite • stroke • thrombus H ydrogen peroxide is used as a hemostatic agent in several neurosurgical centers. Although its exact mechanism has not been clarified, H2O2 is believed to establish hemostasis through its vasoconstrictive properties.22 A few studies, however, have drawn attention to a deleterious effect of the use of H2O2: that is, O2 emboli.6,21,22,36, 38 Another mechanism could be disruption of the blood-brain barrier and aggregation of platelets and neutrophils, leading to thrombus formation and occlusion of the vessel.26,30 Abbreviations used in this paper: BA = basilar artery; H2O2 = hydrogen peroxide; LSA = lenticulostriate artery; MCA = middle cerebral artery; NO = nitric oxide; ROS = reactive oxygen species; 3-NT = 3-nitrotyrosine. 94 • We describe the case of a patient who developed postoperative hemiparesis and dysphasia caused by an LSA stroke after irrigation with H2O2 for hemostasis, although the LSAs were remote from the surgical field and not directly exposed to H2O2. To elucidate the pathophysiological mechanisms of injury caused by H2O2 in vivo, we conducted an experiment in mice in which H2O2 was administered to the brain surface over either an intact or destroyed pial layer and ischemia was reproduced. Illustrative Case This 40-year-old man came to our attention when he experienced generalized seizures for the first time in his life. His neurological examination revealed no pathological findings, but MR imaging revealed a nonenhancing J. Neurosurg. / Vol 110 / January, 2009 Hydrogen peroxide–induced stroke lesion consistent with a low-grade glioma in the left insular region (Fig. 1). The patient was followed up for 4 months, but his seizures continued despite multiple anti­ epi­leptic medications. After detailed preoperative assessment and epilepsy monitoring, surgery was planned to re­sect the tumor. A left extended pterional craniotomy was performed, and dissection of the sylvian fissure revealed the tumor in the insula. The tumor was removed gross totally as described elsewhere.31,32,36,37 All branches of the MCA were meticulously preserved, but the LSAs were not exposed and stayed buried in the putamen. After resection, the tumor cavity was irrigated with 3% H2O2 for ~ 15 seconds to establish hemostasis. As the H2O2 was removed from the cavity by irrigation with saline, multiple bubbles were observed filling the lumen of the polar temporal artery and pulsating along the vessel (see intraoperative video at http://thejns.org). Irrigation with copious amounts of saline immediately removed the H2O2 and no hemodynamic changes were seen during or after this event. Video 1. Intraoperative video showing multiple bubbles in the polar temporal artery in a patient in whom H2O2 was used for hemostasis after tumor removal. The patient awoke with dense right hemiparesis (more pronounced in his arm, 0–2/5) and dysphasia. By his 6-month follow-up examination, the motor strength had returned to normal in his leg and only the distal mus­ cle groups in his right arm remained paretic (2–3/5). His dysphasia had resolved completely. Postoperative MR imaging revealed small, punctate areas of infarction in the distribution of the lenticulostriate vessels (Figs. 2 and 3). Oxygen emboli did not appear in the vascular territory of other branches of the left MCA because of their col­lateral circulation. The LSAs, however, are small end ar­teries without collateral circulation.31,32,36,37 Experimental Study Study Design and Methods We designed an experimental study to evaluate in vivo the mechanism of stroke after irrigation with H2O2. All experiments were done according to local ethics committee regulations. Twenty Swiss albino mice weighing between 22 and 34 g were used for the experiments. The mice were kept at 18–21°C in a 12-hour dark/12-hour light cycle before surgery and anesthetized intraperitoneally with 50 mg/kg ketamine (Ketalar, Parke-Davis) and 5 mg/kg xylazine (Alfazyne, Alfasan). Body temperatures were recorded with a rectal probe and kept at 37 ± 1°C with the aid of a homeothermic temperature control unit (Harvard Apparatus). Four experimental groups of 5 mice each were created and the surgical procedures were performed with the aid of the operating stereomicroscope (Zeiss). In the first group of mice, a unilateral craniectomy with a diameter of 1 cm was made in the right frontal lobe, the dura mater was removed with the pia left intact, and the brain surface was irrigated with normal saline (0.09% NaCl) for 1 minute. In the second group, after the craniectomy the dura mater was removed and the pia left intact, as in the first group. The brain surface was irrigated with 1 ml 3% H2O2 over the intact pial layer for 15 seconds, and then irrigated with normal saline for 1 minute. In the third group of mice, a unilateral frontal craniectomy with resection of the dura mater and peeling of the pia was done. This was followed by a 0.5-cm corticotomy, and the brain surface was irrigated with saline for 1 minute. The fourth group of 5 mice underwent the same procedure as the third, after which the brain surface in the corticotomy area was irrigated for 15 seconds with 1 ml 3% H2O2 and then with saline for 1 minute. After irrigation with either H2O2 or saline, the animals’ hearts were perfused with 4% paraformaldehyde. Fig. 1. Illustrative case. Preoperative axial, coronal, and sagittal T1-weighted MR images of the brain obtained with contrast material revealing a left insular tumor. J. Neurosurg. / Vol 110 / January, 2009 95 M. Mut et al. Fig. 2. Illustrative case. Postoperative axial, coronal, and sagittal T1-weighted MR images obtained with contrast material demonstrating total resection of the left insular tumor. The brain tissues were removed and cryoprotected in 30% sucrose. Serial tangential sections of 20 µm, passing through the craniectomy area, were cut with a cryostat. These sections were stained with 3-NT antibody to detect structures nitrated with peroxynitrite, which causes changes in protein function by nitrating tyrosine residues on the structures. Staining with an antibody against 3-NT indirectly shows peroxynitrite production. Sections were blocked with 5% normal goat serum for 10 minutes; then they were incubated overnight with 3-NT monoclonal antibody (1:100 dilution, Upstate Biotechnology), washed, and incubated with an appropriate secondary antibody for 3 hours (Cy3 Molecular Probes, Invitrogen). The sections were then mounted with Hoechst solution and examined under a Nikon Eclipse E600 fluorescent microscope. Results Tissue Inspection When H2O2 was applied to the cortical surfaces, a vasoconstrictive response was immediately observed in all the arteries and arterioles within the exposed areas. After 15 seconds of exposure to H2O2, multiple bubbles formed almost instantaneously within the lumen of all subpial arteries in the animals in Group 4, in which pial layers had been destroyed and corticotomies had been done. Surprisingly, bubbles were also observed inside the lumen of surface vessels in the animals in Group 2, in which pial layers were intact, immediately after the H2O2 was removed. Application of saline in Groups 1 and 3 did not cause any bubble formation, vasoconstriction, or vasodilation. Histopathological and Immunofluorescence Examination No positive staining for 3-NT immunofluorescence was seen in Group 1 (craniectomy only) or Group 3 (cra­ ni­ectomy only plus corticotomy). Significant immunoreactivity for 3-NT in vessel walls was seen in both groups of H2O2-treated mice (Groups 2 and 4). The 3-NT formation was prominent around the vessels below the site Fig. 3. Axial FLAIR image (A) revealing the punctate areas of infarction in the periventricular white matter (arrowhead). The axial diffusion-weighted images (B and C) reveal the punctate areas of infarction (arrows), which were found in the vascular territory of the LSAs. 96 J. Neurosurg. / Vol 110 / January, 2009 Hydrogen peroxide–induced stroke Fig. 4. Photomicrographs showing that application of H2O2 produces peroxynitrite in the vessel wall. The tissue section was obtained from the right frontal area in a mouse in which the pia was kept intact and the cortical surface was irrigated with H2O2 (Group 2). A: Section with Hoechst staining used as a nuclear marker. B: Section with 3-NT staining in which the peroxynitrite formation was identified. Note the intense perivascular staining encircling a dilated vessel (arrow). Inside the vessel, thrombosis aggregation can be seen (arrowhead). C: The merged images show prominent peroxynitrite formation in the arterial vessel wall. Original magnification × 20. of H2O2 irrigation (Figs. 4 and 5), and there was some positive labeling inside the tissue that clearly reflected the production of peroxynitrite. It is clear that H2O2 application caused prominent peroxynitrite production around the vessels. It must also be noted that 3-NT formation was still prominent in the vessels buried within the neuropil below the intact pial layer, emphasizing the passive and free diffusion of H2O2 through those layers (Fig. 5). The addition of H2O2 and peroxynitrite production resulted in platelet aggregation and acute thrombus formation within the vessels of animals exposed to H2O2. The thrombus formation was demonstrated in sections stained with H & E (Fig. 6). Vascular occlusion is another mechanism responsible for stroke related to H2O2 administration. Discussion Induction of O2 Emboli by H2O2 Hydrogen peroxide can permeate cells relatively well, although some movement through cell membranes may occur via aquaporins.2 Along with its effects on vascular tone, H2O2 can increase the permeability of the endothelium.4,9 A ubiquitous metabolite in living cells, H2O2 is known to decompose into water and O2 by exposure to the family of catalase enzymes. Catalase is found in all cells and acts as a catalyst in the decomposition of H2O2 to avoid production of cell-damaging hydroxyl radicals via reaction with iron ions (Fenton reaction), and thereby regulates the concentration of H2O2 to physiological levels. The production of O2 and water from the decomposition of 2 molecules of H2O2 by catalase is a multistep reaction with iron as a key intermediate.27 Catalase decomposes H2O2 extremely efficiently; up to 200,000 reactions/second. The amount of O2 suddenly liberated can be large enough to cause serious hemodynamic consequences; 1 ml of 3% H2O2 can release 10 ml of O2.10,37,38 There are sporadic reports of O2 embolism after irrigation of the surgical field with H2O2 during neurosurgical procedures such as cervical spine surgery and lumbar discectomy.6,21,22,37,38 In our study, H2O2 caused copious O2 bubbles on the exposed areas of brain (see intraoperative video) after the reaction with catalase in surface cells. More importantly, H2O2 resulted in severe O2 emboli within the surface and also in the deeply embedded arteries and arterioles immediately after its administration to the surface of the brain. Our experimental findings supported the clinical observation that even though Fig. 5. Photomicrographs showing that H2O2 produces peroxynitrite in the vessel wall in which the pial barrier is kept intact. The tissue section was obtained in a mouse frontal lobe just below the area treated with H2O2 and in which the pial barrier was kept intact. A: Section with Hoechst staining used as a nuclear marker. B: Section showing prominent peroxynitrite formation around a vessel and very low-intensity 3-NT staining in the tissue. Note that although strongly positive, the stained vessel is not in direct contact with H2O2 application; peroxynitrite formed around it, possibly due to migration of H2O2 to deep vessels (arrow). C: The merged images show prominent peroxynitrite formation in the arterial vessel wall. Original magnification × 40. J. Neurosurg. / Vol 110 / January, 2009 97 M. Mut et al. Fig. 6. Photomicrographs of tissue sections showing that H2O2 produces acute intravascular thrombus formation in the remote vessels in animals in which the pial barrier is kept intact. Left: Tissue section obtained in a mouse frontal lobe just below the area treated with H2O2. H & E staining shows intravascular thrombus formation and occlusion of vessels. Right: Tissue section obtained in a mouse frontal lobe just below the area treated with normal saline. H & E staining shows no intravascular thrombus formation and no evidence of vessel occlusion. Original magnifications as marked; boxes delineate the enlarged areas on the lower row. the LSAs were not visualized during the surgery, H2O2 diffused through the putamen and reached these arteries with high affinity to produce O2 emboli after its decomposition into O2 and water by the endothelial catalases. Our findings support the suggestion that the use of an excess amount of H2O2 may overwhelm the protective mechanisms of the endothelium, which cannot then cope with the heavy burden of oxidative stress. Excess amounts of H2O2 may diffuse into intravascular compartments through the endothelium and decompose into water and O2 by the action of catalase, in which entrapped free O2 produces the bubbles. The sudden release of excess amounts of O2 cannot be managed, and the O2 itself produces the emboli. Induction of Vasoconstriction and Vascular Occlusion by H2O2 Hydrogen peroxide is commonly used in many neurosurgical centers as a hemostatic agent because of its tentatively identified vasoconstrictive properties,22 but the role of H2O2 in regulating cerebral arterial tone is not completely understood. Exposure to H2O2 may constrict or dilate vessels depending on the species, the segment of the vessel, and the concentration used.1,5,7,20,29 The application of H2O2 dilates the BA in rats.24 Although the endothelial layer is believed to play a role in the dilation of cerebral arteries by H2O2, the exact mechanism in the endothelial cell has not been illuminated. The response to H2O2 has been variously reported to be endothelium-dependent, to be unaffected by the removal of the endothelium, and to occur only in the absence of endothelium.7 98 In canine BAs, H2O2 produced endothelium-dependent contraction in intact vessels,17 but it produced marked re­ laxation in arteries without endothelium.14 When the direct relaxation effects of H2O2 on vascular muscle have been observed, there has been strong evidence that the response is mediated by potassium channels. In vivo studies have consistently described the dilator response of small cerebral arteries and arterioles to H2O2.19,28,34,35 Very high concentrations of H2O2 can produce vasoconstriction followed by vasodilation.19 Even more perplexing is the fact that H2O2 application to mouse brain produced peroxynitrite around the ves­sel walls (Figs. 4 and 5). Hydrogen peroxide is a toxic ROS that is kept at physiological levels via degradation by catalase and the glutathione/glutathione peroxidase system.25 The ROS may convert to each other through a series of reactions in the presence of nitric oxide synthase, iron, copper, glutathione peroxidase, superoxide dismutase, catalase, and nicotinamide adenine dinucleo­ tide phos­phate (reduced form) oxidase.7,23,33 In the presence of excess amounts of H2O2, which the protective mechanisms of the endothelium cannot manage, the reaction of NO with H2O2 generates peroxynitrite via multiple reactions; a high amount of O2 inside the vessel can produce superoxide. Production of superoxide exceeding the limits of elimination enters the reaction with NO produced in endothelium, causing peroxynitrite formation.18 Additionally, hydroxyl radicals generated by the Fenton reaction or H2O2 itself react with NO in the presence of iron to produce peroxynitrite.8,11,15,16 The combination of NO and H2O2 was particularly cytotoxic and mediated by generated NO radicals. Among the NO radicals, peroxynitrite is a very potent and destructive oxidant and may produce some of its deleterious effects by nitrating tyrosine residues on target proteins. The number of studies showing evidence that peroxynitrite is produced in the brain or cerebral blood vessels under various pathophysiological conditions continues to increase.12,13 Nevertheless, relatively few investigators have examined what direct effects peroxynitrite may have on vascular tone. Wei and colleagues35 were the first to show that relatively low concentrations of peroxynitrite dilate cerebral arterioles in vivo. The pharmacological profile they obtained suggested that this response was mediated by adenosine 5ʹ-triphosphate–sensitive potassium channels. In contrast, other studies have reported that peroxynitrite contracts cerebral arteries in vitro via an inhibitory effect of the basal activity of calcium-activated potassium channels.3 In our study in mice, in which we used a high concentration of H2O2 administered directly to the brain and cerebral vasculature, severe vasoconstriction followed by vasodilation was observed. This response might be specifically mediated by the production of peroxynitrite in the endothelial cells. Another deleterious effect of H2O2 and peroxynitrite is blood-brain barrier breakdown and intravascular aggregates of platelets and neutrophils and thrombus formation. Although this mechanism has not been mentioned in the surgical literature, platelet aggregation could be another series of events leading to acute thrombus formation and hemostasis after application of H2O2. J. Neurosurg. / Vol 110 / January, 2009 Hydrogen peroxide–induced stroke Hydrogen peroxide has an affinity to vessel walls. In our experimental study, even when the pial layers were kept intact, H2O2 diffused through the neuropil and reached the vessel walls to produce its ROS and resultant vasoactive responses that may be the cause of ischemia. Besides the vasoactive responses and platelet aggregation, which are most likely created by the ROS and nitrous species, the abundant existence of O2 bubbles within the vessel lumen may result in stroke. In considering significant adverse effects due to H2O2 irrigation, formation of O2 emboli on the vessel wall as well as injury to the wall by ROS should be kept in mind as underlying mechanisms. Conclusions Hydrogen peroxide is used in neurosurgical practice as a hemostatic agent. However, it may cause serious complications due to cerebral or systemic O2 emboli and cerebral vasoconstriction. Because H2O2 can easily pass through the intact pial layer and penetrate cerebral vessels, its use intradurally for hemostasis should be prohibited. Hydrogen peroxide can cause severe embolic strokes. In fact, its use should be strictly limited to clearly open wounds in which no possibility of gas entrapment can occur. The only indication for H2O2 as a hemostatic agent may be in extradural skull base surgery, but even so, this substance should be cautiously administered with immediate irrigation and removal. Disclosure The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper. References 1. 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Wei EP, Kontos HA: H2O2 and endothelium-dependent cerebral arteriolar dilation. Hypertension 16:162–169, 1990 35. Wei EP, Kontos HA, Beckman JS: Mechanisms of cerebral vasodilation by superoxide, hydrogen peroxide, and peroxynitrite. Am J Physiol 271:H1262–H1266, 1996 36. Yaşargil MG: The central zone in white matter (external and internal capsules), in Microneurosurgery. Stuttgart: Thieme, Vol 4A, 1994, pp 65–73 37. Yaşargil MG: Microneurosurgery. Stuttgart: Thieme, Vol 4B, 1996, pp 262–276 38. Zimmerman GA, Lipow KI: Pneumocephalus with neurologi- 100 cal deficit from hydrogen peroxide irrigation. Case illustration. J Neurosurg 100:1122, 2004 Manuscript submitted September 6, 2007. Accepted March 10, 2008. Please include this information when citing this paper: published online October 17, 2008; DOI: 10.3171/2008.3.17434. Supplemental online information: Video: To view with Windows Media Player and a broadband connection go to: http://mfile.akamai.com/21490/wmv/digitalwbc. download.akamai.com/21492/wm.digitalsource-na-regional/17434_ Mut.asx or to view with RealPlayer go to: http://mfile.akamai.com/ 21489/rm/digitalwbc.download.akamai.com/21492/real.digitalsource-na-regional/17434_Mut.ram. Address correspondence to: Melike Mut, M.D., Ph.D., Department of Neurosurgery, Hacettepe University, Beyin Cerrahisi 1. Kat Sihhiye, 06100 Ankara, Turkey. email:melikem@hacettepe.edu.tr or mm2ee@virginia.edu. J. Neurosurg. / Vol 110 / January, 2009