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Nuclear factor-κB and apoptosis in patients with intracerebral hemorrhage
Journal of Clinical Neuroscience 18 (2011) 1392–1395
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Laboratory Study
Nuclear factor-jB and apoptosis in patients with intracerebral hemorrhage Ya-Xian Wang a, Ai Yan b, Zhi-Hong Ma c, Zhuang Wang a, Bing Zhang a, Jin-liang Ping d, Jin-Shui Zhu a, Yue Zhou b, Licheng Dai c,⇑ a
Department of Neurology, Huzhou Central Hospital, Huzhou, Zhejiang Province, China Department of Neurosurgery, Huzhou Central Hospital, Huzhou, Zhejiang Province, China c Huzhou Key Laboratory of Molecular Medicine, Affiliated Central Hospital of Huzhou Teachers College, Huzhou 313000, Zhejiang Province, China d Department of Pathology, Huzhou Central Hospital, Huzhou, Zhejiang Province, China b
a r t i c l e
i n f o
Article history: Received 6 September 2010 Accepted 17 November 2010
Keywords: Apoptosis Inflammatory cytokines Intracerebral hemorrhage Nuclear factor-jB
a b s t r a c t Studies using rat models have indicated that neuronal apoptosis is involved in the pathogenesis of intracerebral hemorrhage (ICH); however, the mechanism by which apoptosis occurs is unclear. In the present study, we aimed to quantify the number of nuclear factor-jB (NF-jB)-positive cells and apoptotic cells in specimens of middle temporal gyrus taken from 46 human subjects with hypertensive ICH. We also investigated the roles that intercellular adhesion molecule-1 (ICAM-1) and interleukin (IL)-1b play in apoptosis following ICH. At about 24 hours after ICH, some neurons exhibited nuclear swelling and incomplete cellular structures were visible. The mean percentage of apoptotic cells was 39.28 ± 21.83% at 49–72 hours after ICH. NF-jB immunoreactivity varied with time after ICH: the number of immunostained neurons increased during the 2–6 hours after ICH, and reached a maximum at 7–48 hours. The number of IL-1b-immunostained neurons reached a maximum at 2–6 hours after ICH. The number of ICAM-1-immunostained neurons increased during the 48 hours after ICH and reached a maximum at 49–72 hours. These observations indicate that apoptosis has a major role in pathological cell death after ICH and that activation of NF-jB is positively related to the progress of apoptosis. Additionally, activation of ICAM-1 and IL-1b seem to be involved in apoptosis after ICH. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Intracerebral hemorrhage (ICH) is one of the most devastating types of stroke and is associated with high mortality and morbidity.1–5 Around 50% of ICH patients die within the first 48 hours and fewer than 20% of survivors return to the normal activities of daily living.6 However, at present, there are no pharmacological agents available for use in clinical practice. Thus, experimental studies are required to better understand the events leading to brain injury following ICH. Hematoma is known to cause vasogenic edema, breakdown of the blood–brain barrier and glial cell death.7–9 Cellular death can be subdivided two types: necrosis and apoptosis.10 Recent studies using animal models have shown that cell death in the perihematoma region may be mediated by apoptosis.11 In order to better understand the role of apoptosis in ICH, in the present study we aimed to investigate the relationship between neuronal injury and apoptosis in ICH. Nuclear factor-jB (NF-jB) is a ubiquitous transcription factor that is extremely sensitive to oxidative stress in perihematomal brain regions after ICH. NF-jB is also an important mediator of ⇑ Corresponding author. Tel./fax: +86 572 2033020. E-mail address: [email protected] (L. Dai). 0967-5868/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jocn.2010.11.039
the coordinated induction of central nervous system genes, including proinflammatory cytokines such as interleukin (IL)-1b.12–15 IL1b has a central role in the regulation of immune and inflammatory responses. Because inflammatory responses may be involved in cell damage and death after ICH, NF-jB is likely to play a critical role in cell death, especially apoptosis. In the present study, we aimed to measure the extent of apoptosis and activation of NFjB in patients with ICH. Our aim was to develop a better understanding of the role of NF-jB in the pathological processes of cell damage and death after ICH. 2. Patients and methods 2.1. Patients and specimens Tissue samples were collected from 46 patients with hypertensive ICH between January 2006 and June 2007. All patients provided written consent. The diagnosis of ICH was based on the Diagnostic Criteria of Hypertensive Cerebral Hemorrhage (established at the Fourth National Cerebrovascular Disease Conference).16 ICH in the basal ganglia was confirmed in all patients by cranial CT scan or MRI. Exclusion criteria were: bleeding resulting from traumatic brain injury, cerebral hemorrhage caused by
Y.-X. Wang et al. / Journal of Clinical Neuroscience 18 (2011) 1392–1395
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Fig. 1. Hematoxylin and eosin-stained sections of temporal gyrus showing morphological changes in cells at around 24 hours after intracerebral hemorrhage. Con = control (no intracerebral hemorrhage), 3# = patient 3, 8# = patient 8, 10# = patient 10. Nuclear swelling and incomplete cellular structures are seen in some neurons in the patient specimens, but not in that from the controls (original magnification 100).
anticoagulants, cancer, cerebral vascular malformation hemorrhage, hemorrhagic infarction, liver and kidney disease with severe heart failure, and severe pulmonary infection. Based on the time since ICH, patients were divided into the following groups: the ultra-early onset (<6 hours) operation group, the early (7–48 hours) group, and the extension (49–72 hours) group (see Table 2). All patients underwent neurosurgical evacuation procedures as follows: a straight 5-cm incision was made below the Sylvian fissure, a skull bone window 2.5–3.0 cm in diameter was created, and the cerebral dura mater was cut in a stellate fashion to expose the middle temporal gyrus and parts of the superior and inferior temporal gyrus. Bipolar coagulation was used to achieve hemostasis in the cortical vessels of the middle temporal gyrus. Parts of the temporal gyrus cortex were removed until the hematoma cavity was reached, and the blood clot was gently sucked out. Intracranial decompression was also performed on patients with large hematomas, significant brain swelling and midline shift, or brain herniation, by removal of the temporal lobe anterior to the vein of Labbé. A small amount of tissue from the middle temporal gyrus around the hematoma was obtained during surgical resection. The tissue samples were immediately immersed in 4% paraformaldehyde. Normal brain tissues were used as controls, which were taken from five patients undergoing resection of pathologically confirmed aneurysm. The control specimens were taken from the edge of the aneurysm. The study methodology was approved by Huzhou Central Hospital Ethics Committee. 2.2. Hematoxylin and eosin staining and TUNEL assays The formalin-fixed, paraffin-embedded specimens were sectioned at 5 lm and stained with hematoxylin and eosin. To stain cells undergoing apoptosis, a terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labeling (TUNEL) assay was performed according to the instructions provided by the manufacturer (ApopTag peroxidase in situ apoptosis detection kit; Intergen Co., Purchase, NY, USA). TUNEL is a common method for detecting DNA fragmentation resulting from apoptotic signaling cascades. The assay relies on the presence of nicks in the DNA which can be identified by terminal deoxynucleotidyl transferase, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. It may also label cells that have suffered severe DNA damage. Tissue sections were treated with proteinase K (20 lg/mL) for 20 minutes and washed three times with distilled water. The sections were then treated with hydrogen peroxide for 10 minutes at room temperature, rinsed twice with phosphate-buffered saline, and rinsed with deoxyribonuclease solution for 10 minutes, followed by an equalization buffer. Terminal deoxynucleotidyl transferase reaction buffer was added, and the sections were incubated for 1 hour at 37 °C. The slides were rinsed three times after application of stop wash buffer, and antidigoxigenin conjugate was applied for 30 minutes. The
slides were washed four times with phosphate-buffered saline (PBS) for 5 minutes, and diaminobenzidine was added. 2.3. Immunohistochemical analysis Immunohistochemical analysis was carried out using the following primary antibodies: NF-jB (#4764; Cell Signaling Technology, Beverly, MA, USA), IL-1b (#8900; Cell Signaling Technology), and ICAM-1 (#4915; Cell Signaling Technology). The tissue sections were dewaxed and rehydrated, rinsed with distilled water and PBS, quenched with 3% H2O2, and then exposed to primary antibodies and incubated at 4 °C overnight. Sections were washed with Triton–PBS, incubated in biotinylated mouse anti-rabbit immunoglobulin G (IgG) or rabbit anti-goat IgG for 1 hour at room temperature, washed again, and incubated with streptavidin–peroxidase for 30 minutes at room temperature. Finally, the sections were stained with diaminobenzidine–H2O2 solution, washed, dehydrated in graded ethanol, immersed in xylene, and covered with a cover slip. 2.4. Statistical analyses Student’s t-test and analysis of variance (ANOVA) were used to evaluate the results of the immunohistochemical and TUNEL analysis. The correlation between the level of apoptosis and activation of NF-jB was analyzed using Pearson’s correlation coefficient. Statistical significance was set at p < 0.05. 3. Results 3.1. Apoptosis is involved in intracerebral hemorrhage When tissue specimen sections obtained 24 hours after ICH were examined, some neurons exhibited nuclear swelling and had incomplete cellular structures. Inflammatory cells, including mononuclear cells and neutrophils, were evident. In the control group, few morphologic changes were observed (Fig. 1). The TUNEL assay was used to stain apoptotic cells in sections from 46 ICH patients and control patients also. Apoptosis was evident in 42 of the 46 surgical specimens. The mean percentage of apoptotic cells among patients was 39.28 ± 21.83% at 49–72 hours. In more than 10 patients, more than half of all cells were apoptotic (Table 1, Supplementary Fig. 1). 3.2. Correlation between degree of apoptosis and activation of nuclear factor-jB Pearson’s correlation analysis revealed strong linear correlation between degree of apoptosis and activation of NF-jD in all 42 patients with ICH who had evidence of apoptosis in their surgical specimen (R = 0.7399; p < 0.0001; Fig. 2).
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Y.-X. Wang et al. / Journal of Clinical Neuroscience 18 (2011) 1392–1395
Table 1 Demographic and clinical features of patients with hypertensive intracerebral hemorrhage and controls, including the percentage of apoptotic cells in the middle temporal gyrus Patient no.
Age (years)
Sex
MAP (mmHg)
Volume (cm3)
Apoptotic cells (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Control Control Control Control Control
35 48 51 56 49 61 70 63 57 61 49 63 54 39 41 63 61 56 55 71 73 78 64 45 44 72 61 62 69 38 56 62 61 67 46 55 57 68 67 65 74 77 46 64 65 57 48 61 66 50 55
M M F M M M F F M M F M M M F M M M F M M M M F M M M F M M F F M F M M M F M M M F F F M M M F M M F
165 100 112 123 116 132 117 113 163 118 132 154 177 123 110 118 112 126 164 154 123 112 132 177 112 123 143 100 132 124 154 165 121 112 117 110 164 135 162 102 125 99 120 138 119 134 154 119 124 120 132
45 66 54 74 112 47 48 67 35 79 83 39 108 56 58 72 57 61 56 93 48 41 36 63 35 64 53 73 35 74 43 46 46 42 74 64 34 78 96 65 75 46 39 111 45 67
41 37 48 20 60 45 82 55 70 40 10 15 45 20 15 42 64 30 0 54 12 74 63 53 0 36 56 72 0 35 61 45 34 25 35 35 90 25 64 0 24 54 37 43 37 57 0 8 6 10 5
MAP = mean arterial pressure, Volume = volume of the intracerebral hematoma.
3.3. Expression of nuclear factor-jB, interleukin-1b and intercellular adhesion molecule-1 On the injured side of the middle temporal gyrus, nuclear factor-jB immunoreactivity varied with time after ICH: the number of immunostained neurons was higher than control at 2–6 hours after ICH, reached a maximum at 7–48 hours after ICH (p < 0.01, compared with control group), then had decreased slightly at 49–72 hours after ICH (Table 2). The number of IL-1b-immunostained neurons reached a maximum at 2–6 hours after ICH (p < 0.01, compared with the control group; Table 2). The number of ICAM-1-immunostained neurons was higher than control at 2– 48 hours after ICH and reached a maximum at 49–72 hours after ICH (p < 0.01, compared with control group and earlier group; Table 2, Supplementary Fig. 2).
4. Discussion Intact, injured and necrotic cells can be seen in regions close to and distant from the hematoma in a rabbit model of ICH.17 When Qureshi et al. collected tissue specimens from boundary tissue after removing intracerebral hematomas in 14 patients, they found that 38% of cells in the perihematoma region had undergone apoptotic changes. However, in that study the number of patients was small.18 In the present study, we observed and assessed apoptosis on the basis of the morphological and staining characteristics of cells in 46 patients with ICH. Apoptosis was evident in 42 of the 46 surgical specimens available for analysis. In more than 10 patients, more than half of all cells were apoptotic. Interestingly, four patients had no apoptotic cells. There was no association between the percentage apoptotic cells and patient age or hematoma volume. The transcription factor NF-jB is a regulator of cell death and survival. Following experimental ICH in rats, Hickenbottom et al. found a 1.8–2.5-fold increase in NF-jB levels in ipsilateral perihematoma samples obtained 4 days after ICH.19 In the present study, activation of NF-jB varied with time after ICH. The number of immunostained neurons reached a maximum at 7–48 hours after ICH (p < 0.01, compared with control group). Furthermore, an increase in the number of immunoreactive neurons was associated with the degree of apoptosis. These findings suggest a relationship between NF-jB and the pathobiological mechanism of apoptotic cell death after ICH. Activation of NF-jB leads to rapid and coordinated induction of proteolytic enzymes and release of proinflammatory cytokines and chemokines. To further understand the potential implications of NF-jB activation, it is imperative to characterize the expression of proinflammatory cytokine and chemokine gene product changes after ICH. The proinflammatory contribution of IL-1b in various brain injury models, in neurodegeneration and in increased blood–brain-barrier permeability and edema has been well described.20–22 We found that the number of IL-1b-immunostained neurons reached a maximum at 2–6 hours after ICH (p < 0.01, compared with control group; Table 2). This means that expression of IL-1b may be regulated by the activation of NF-jB. ICAM-1 is an endothelial- and leukocyte-associated transmembrane protein long known for its importance in stabilizing cell–cell interactions and facilitating leukocyte endothelial transmigration. Because of these associations with immune responses, many researchers have hypothesized that ICAM-1 could function in signal transduction of immune reactions. In the present study, we showed that the number of ICAM-1-immunostained neurons was higher relative to control at 2–48 hours after ICH and reached a maximum at 49– 72 hours after ICH (p < 0.01 compared with control group). We hypothesize that ICAM-1 might act as a mediator of NF-jB proapoptotic activity after ICH. In the present study, we found that apoptosis plays an important role in cell death after ICH. That activation of NF-jB was positively associated with the progress of apoptosis indicates that NF-jB could be a vital mediator of apoptosis transduction. Table 2 Expression of nuclear factor-jB (NF-jB), interleukin-1b (IL-1b), and intercellular adhesion molecule-1 (ICAM-1) in sections of temporal gyrus from patients with hypertensive intracerebral hemorrhage (ICH) (n = 46) and controls (n = 5) Time since ICH (hours)
n
NF-jB
IL-1b
ICAM-1
2–6 7–48 49–72 Control
15 18 13 5
12.5 ± 3.9⁄ 30 ± 2.3⁄⁄ 24 ± 4.6⁄⁄ 6.1 ± 1.2
25.4 ± 7.2⁄⁄ 12.1 ± 3.6⁄ 13.0 ± 0.3⁄ 8.8.0 ± 1.1
2.3 ± 0.2⁄⁄ 10.7 ± 2.4⁄⁄ 17.6 ± 1.6⁄⁄ 0.6 ± 0.1
Data are mean ± standard deviation. ⁄p < 0.05 compared with control group, p < 0.01 compared with control group.
⁄⁄
Y.-X. Wang et al. / Journal of Clinical Neuroscience 18 (2011) 1392–1395
Fig. 2. Correlation between percentage of apoptotic cells and percentage nuclear factor-jB (NF-jB)-positive cells (%) in the middle temporal gyrus of 42 patients with intracerebral hemorrhage. There was a positive linear relationship between the two variables.
Activation of IL-1b and ICAM-1 shows that NF-jB may be involved in apoptosis via activating inflammatory cytokines. Our data suggest that NF-jB could be a diagnostic or therapeutic target for ICH. Acknowledgement This study was supported by the Scientific Research Fund of Huzhou City (no. 2006YZ01). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jocn.2010.11.039. References 1. Kase CS, Caplan LR. Intracerebral hemorrhage. Newton, MA: ButterworthHeinemann; 1994. 2. Qureshi AI, Tuhrim S, Broderick JP, et al. Spontaneous intracerebral hemorrhage. N Engl J Med 2001;344:1450–60.
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3. Mayer SA, Sacco RL, Shi T, et al. Neurologic deterioration in noncomatose patients with supratentorial intracerebral hemorrhage. Neurology 1994;44:1379–84. 4. Mendelow AD. Spontaneous intracerebral haemorrhage. J Neurol Neurosurg Psychiatry 1991;54:193–5. 5. Broderick JP, Brott TG, Duldner JE, et al. Volume of intracerebral hemorrhage: a powerful and easy-to-use predictor of 30-day mortality. Stroke 1993;24:987–93. 6. Wagner KR. Modeling intracerebal hemorrhage: glutamate, nuclear factorkappaB signaling and cytokines. Stroke 2007;38:753–8. 7. Thatte U, Dahanukar S. Apoptosis: clinical relevance and pharmacological manipulation. Drugs 1997;54:511–32. 8. Wisniewski H. The pathogenesis of some cases of cerebral hemorrhage: a morphological study of brain distant from the hemorrhage. Acta Med Pol 1961;2:379–90. 9. Chesney JA, Kondoh BA, Conrad BS, et al. Collagenase-induced intrastriatal hemorrhage in rats results in long-term locomotor deficits. Stroke 1995;26:312–7. 10. Wagner KR, Xi G, Hua Y, et al. Lobar intracerebral hemorrhage model in pigs: rapid edema development in perihematomal white matter. Stroke 1996;27:490–7. 11. Qin ZH, Wang Y, Nakai M, et al. Nuclear factor-kappa B contributes to excitotoxin-induced apoptosis in rat striatum. Mol Pharmacol 1998;53:33–42. 12. Van Antwerp DJ, Martin SJ, Kafri T, et al. Suppression of TNF-a-induced apoptosis by NF-jB. Science 1996;274:787–9. 13. Yin MJ, Yamamoto Y, Gaynor RB. The anti-inflammatory agents aspirin and salicylate inhibit the activity of I(kappa)B kinase-beta. Nature 1998;396:77–80. 14. Schmitz ML, Baeuerle PA. Major transcription factors: multi-step activation of NF-jB/rel transcription factors. Immunobiology 1995;193:116–27. 15. Deinsberger W, Vogel J, Kuschinsky W, et al. Experimental intracerebral hemorrhage: description of a double injection model in rats. Neurol Res 1996;5:475–7. 16. Wang XD. Diagnostic gist of various cerebrovascular diseases. Zhonghua Shenjingke Zazhi 1996;29:379–80. 17. Qureshi AI, Ling GS, Khan J, et al. Quantitative analysis of injured, necrotic, and apoptotic cells in a new experimental model of intracerebral hemorrhage. Crit Care Med 2001;29:152–7. 18. Qureshi AI, Suri MF, Ostrow PT, et al. Apoptosis as a form of cell death in intracerebral hemorrhage. Neurosurgery 2003;52:1041–8. 19. Wagner KR, Dean C, Beiler S, et al. Plasma infusions into porcine cerebral white matter induces early edema, oxidative stress, pro-inflammatory cytokine gene expression and DNA fragmentation: Implications for white matter injury with increased blood-brain barrier permeability. Curr Neurovasc Res 2005;2:149–55. 20. Matsushita K, Meng W, Wang X, et al. Evidence for apoptosis after intercerebral hemorrhage in rat striatum. J Cereb Blood Flow Metab 2000;20:396–404. 21. Wu J, Hua Y, Keep RF, et al. Oxidative brain injury from extravasated erythrocytes after intracerebral hemorrhage. Brain Res 2002;953:45–52. 22. Felberg RA, Grotta JC, Shirzadi AL, et al. Cell death in experimental intracerebral hemorrhage: the ‘‘black hole’’ model of hemorrhagic damage. Ann Neurol 2002;51:517–24.
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Laboratory Study
Nuclear factor-jB and apoptosis in patients with intracerebral hemorrhage Ya-Xian Wang a, Ai Yan b, Zhi-Hong Ma c, Zhuang Wang a, Bing Zhang a, Jin-liang Ping d, Jin-Shui Zhu a, Yue Zhou b, Licheng Dai c,⇑ a
Department of Neurology, Huzhou Central Hospital, Huzhou, Zhejiang Province, China Department of Neurosurgery, Huzhou Central Hospital, Huzhou, Zhejiang Province, China c Huzhou Key Laboratory of Molecular Medicine, Affiliated Central Hospital of Huzhou Teachers College, Huzhou 313000, Zhejiang Province, China d Department of Pathology, Huzhou Central Hospital, Huzhou, Zhejiang Province, China b
a r t i c l e
i n f o
Article history: Received 6 September 2010 Accepted 17 November 2010
Keywords: Apoptosis Inflammatory cytokines Intracerebral hemorrhage Nuclear factor-jB
a b s t r a c t Studies using rat models have indicated that neuronal apoptosis is involved in the pathogenesis of intracerebral hemorrhage (ICH); however, the mechanism by which apoptosis occurs is unclear. In the present study, we aimed to quantify the number of nuclear factor-jB (NF-jB)-positive cells and apoptotic cells in specimens of middle temporal gyrus taken from 46 human subjects with hypertensive ICH. We also investigated the roles that intercellular adhesion molecule-1 (ICAM-1) and interleukin (IL)-1b play in apoptosis following ICH. At about 24 hours after ICH, some neurons exhibited nuclear swelling and incomplete cellular structures were visible. The mean percentage of apoptotic cells was 39.28 ± 21.83% at 49–72 hours after ICH. NF-jB immunoreactivity varied with time after ICH: the number of immunostained neurons increased during the 2–6 hours after ICH, and reached a maximum at 7–48 hours. The number of IL-1b-immunostained neurons reached a maximum at 2–6 hours after ICH. The number of ICAM-1-immunostained neurons increased during the 48 hours after ICH and reached a maximum at 49–72 hours. These observations indicate that apoptosis has a major role in pathological cell death after ICH and that activation of NF-jB is positively related to the progress of apoptosis. Additionally, activation of ICAM-1 and IL-1b seem to be involved in apoptosis after ICH. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Intracerebral hemorrhage (ICH) is one of the most devastating types of stroke and is associated with high mortality and morbidity.1–5 Around 50% of ICH patients die within the first 48 hours and fewer than 20% of survivors return to the normal activities of daily living.6 However, at present, there are no pharmacological agents available for use in clinical practice. Thus, experimental studies are required to better understand the events leading to brain injury following ICH. Hematoma is known to cause vasogenic edema, breakdown of the blood–brain barrier and glial cell death.7–9 Cellular death can be subdivided two types: necrosis and apoptosis.10 Recent studies using animal models have shown that cell death in the perihematoma region may be mediated by apoptosis.11 In order to better understand the role of apoptosis in ICH, in the present study we aimed to investigate the relationship between neuronal injury and apoptosis in ICH. Nuclear factor-jB (NF-jB) is a ubiquitous transcription factor that is extremely sensitive to oxidative stress in perihematomal brain regions after ICH. NF-jB is also an important mediator of ⇑ Corresponding author. Tel./fax: +86 572 2033020. E-mail address: [email protected] (L. Dai). 0967-5868/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jocn.2010.11.039
the coordinated induction of central nervous system genes, including proinflammatory cytokines such as interleukin (IL)-1b.12–15 IL1b has a central role in the regulation of immune and inflammatory responses. Because inflammatory responses may be involved in cell damage and death after ICH, NF-jB is likely to play a critical role in cell death, especially apoptosis. In the present study, we aimed to measure the extent of apoptosis and activation of NFjB in patients with ICH. Our aim was to develop a better understanding of the role of NF-jB in the pathological processes of cell damage and death after ICH. 2. Patients and methods 2.1. Patients and specimens Tissue samples were collected from 46 patients with hypertensive ICH between January 2006 and June 2007. All patients provided written consent. The diagnosis of ICH was based on the Diagnostic Criteria of Hypertensive Cerebral Hemorrhage (established at the Fourth National Cerebrovascular Disease Conference).16 ICH in the basal ganglia was confirmed in all patients by cranial CT scan or MRI. Exclusion criteria were: bleeding resulting from traumatic brain injury, cerebral hemorrhage caused by
Y.-X. Wang et al. / Journal of Clinical Neuroscience 18 (2011) 1392–1395
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Fig. 1. Hematoxylin and eosin-stained sections of temporal gyrus showing morphological changes in cells at around 24 hours after intracerebral hemorrhage. Con = control (no intracerebral hemorrhage), 3# = patient 3, 8# = patient 8, 10# = patient 10. Nuclear swelling and incomplete cellular structures are seen in some neurons in the patient specimens, but not in that from the controls (original magnification 100).
anticoagulants, cancer, cerebral vascular malformation hemorrhage, hemorrhagic infarction, liver and kidney disease with severe heart failure, and severe pulmonary infection. Based on the time since ICH, patients were divided into the following groups: the ultra-early onset (<6 hours) operation group, the early (7–48 hours) group, and the extension (49–72 hours) group (see Table 2). All patients underwent neurosurgical evacuation procedures as follows: a straight 5-cm incision was made below the Sylvian fissure, a skull bone window 2.5–3.0 cm in diameter was created, and the cerebral dura mater was cut in a stellate fashion to expose the middle temporal gyrus and parts of the superior and inferior temporal gyrus. Bipolar coagulation was used to achieve hemostasis in the cortical vessels of the middle temporal gyrus. Parts of the temporal gyrus cortex were removed until the hematoma cavity was reached, and the blood clot was gently sucked out. Intracranial decompression was also performed on patients with large hematomas, significant brain swelling and midline shift, or brain herniation, by removal of the temporal lobe anterior to the vein of Labbé. A small amount of tissue from the middle temporal gyrus around the hematoma was obtained during surgical resection. The tissue samples were immediately immersed in 4% paraformaldehyde. Normal brain tissues were used as controls, which were taken from five patients undergoing resection of pathologically confirmed aneurysm. The control specimens were taken from the edge of the aneurysm. The study methodology was approved by Huzhou Central Hospital Ethics Committee. 2.2. Hematoxylin and eosin staining and TUNEL assays The formalin-fixed, paraffin-embedded specimens were sectioned at 5 lm and stained with hematoxylin and eosin. To stain cells undergoing apoptosis, a terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labeling (TUNEL) assay was performed according to the instructions provided by the manufacturer (ApopTag peroxidase in situ apoptosis detection kit; Intergen Co., Purchase, NY, USA). TUNEL is a common method for detecting DNA fragmentation resulting from apoptotic signaling cascades. The assay relies on the presence of nicks in the DNA which can be identified by terminal deoxynucleotidyl transferase, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. It may also label cells that have suffered severe DNA damage. Tissue sections were treated with proteinase K (20 lg/mL) for 20 minutes and washed three times with distilled water. The sections were then treated with hydrogen peroxide for 10 minutes at room temperature, rinsed twice with phosphate-buffered saline, and rinsed with deoxyribonuclease solution for 10 minutes, followed by an equalization buffer. Terminal deoxynucleotidyl transferase reaction buffer was added, and the sections were incubated for 1 hour at 37 °C. The slides were rinsed three times after application of stop wash buffer, and antidigoxigenin conjugate was applied for 30 minutes. The
slides were washed four times with phosphate-buffered saline (PBS) for 5 minutes, and diaminobenzidine was added. 2.3. Immunohistochemical analysis Immunohistochemical analysis was carried out using the following primary antibodies: NF-jB (#4764; Cell Signaling Technology, Beverly, MA, USA), IL-1b (#8900; Cell Signaling Technology), and ICAM-1 (#4915; Cell Signaling Technology). The tissue sections were dewaxed and rehydrated, rinsed with distilled water and PBS, quenched with 3% H2O2, and then exposed to primary antibodies and incubated at 4 °C overnight. Sections were washed with Triton–PBS, incubated in biotinylated mouse anti-rabbit immunoglobulin G (IgG) or rabbit anti-goat IgG for 1 hour at room temperature, washed again, and incubated with streptavidin–peroxidase for 30 minutes at room temperature. Finally, the sections were stained with diaminobenzidine–H2O2 solution, washed, dehydrated in graded ethanol, immersed in xylene, and covered with a cover slip. 2.4. Statistical analyses Student’s t-test and analysis of variance (ANOVA) were used to evaluate the results of the immunohistochemical and TUNEL analysis. The correlation between the level of apoptosis and activation of NF-jB was analyzed using Pearson’s correlation coefficient. Statistical significance was set at p < 0.05. 3. Results 3.1. Apoptosis is involved in intracerebral hemorrhage When tissue specimen sections obtained 24 hours after ICH were examined, some neurons exhibited nuclear swelling and had incomplete cellular structures. Inflammatory cells, including mononuclear cells and neutrophils, were evident. In the control group, few morphologic changes were observed (Fig. 1). The TUNEL assay was used to stain apoptotic cells in sections from 46 ICH patients and control patients also. Apoptosis was evident in 42 of the 46 surgical specimens. The mean percentage of apoptotic cells among patients was 39.28 ± 21.83% at 49–72 hours. In more than 10 patients, more than half of all cells were apoptotic (Table 1, Supplementary Fig. 1). 3.2. Correlation between degree of apoptosis and activation of nuclear factor-jB Pearson’s correlation analysis revealed strong linear correlation between degree of apoptosis and activation of NF-jD in all 42 patients with ICH who had evidence of apoptosis in their surgical specimen (R = 0.7399; p < 0.0001; Fig. 2).
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Y.-X. Wang et al. / Journal of Clinical Neuroscience 18 (2011) 1392–1395
Table 1 Demographic and clinical features of patients with hypertensive intracerebral hemorrhage and controls, including the percentage of apoptotic cells in the middle temporal gyrus Patient no.
Age (years)
Sex
MAP (mmHg)
Volume (cm3)
Apoptotic cells (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Control Control Control Control Control
35 48 51 56 49 61 70 63 57 61 49 63 54 39 41 63 61 56 55 71 73 78 64 45 44 72 61 62 69 38 56 62 61 67 46 55 57 68 67 65 74 77 46 64 65 57 48 61 66 50 55
M M F M M M F F M M F M M M F M M M F M M M M F M M M F M M F F M F M M M F M M M F F F M M M F M M F
165 100 112 123 116 132 117 113 163 118 132 154 177 123 110 118 112 126 164 154 123 112 132 177 112 123 143 100 132 124 154 165 121 112 117 110 164 135 162 102 125 99 120 138 119 134 154 119 124 120 132
45 66 54 74 112 47 48 67 35 79 83 39 108 56 58 72 57 61 56 93 48 41 36 63 35 64 53 73 35 74 43 46 46 42 74 64 34 78 96 65 75 46 39 111 45 67
41 37 48 20 60 45 82 55 70 40 10 15 45 20 15 42 64 30 0 54 12 74 63 53 0 36 56 72 0 35 61 45 34 25 35 35 90 25 64 0 24 54 37 43 37 57 0 8 6 10 5
MAP = mean arterial pressure, Volume = volume of the intracerebral hematoma.
3.3. Expression of nuclear factor-jB, interleukin-1b and intercellular adhesion molecule-1 On the injured side of the middle temporal gyrus, nuclear factor-jB immunoreactivity varied with time after ICH: the number of immunostained neurons was higher than control at 2–6 hours after ICH, reached a maximum at 7–48 hours after ICH (p < 0.01, compared with control group), then had decreased slightly at 49–72 hours after ICH (Table 2). The number of IL-1b-immunostained neurons reached a maximum at 2–6 hours after ICH (p < 0.01, compared with the control group; Table 2). The number of ICAM-1-immunostained neurons was higher than control at 2– 48 hours after ICH and reached a maximum at 49–72 hours after ICH (p < 0.01, compared with control group and earlier group; Table 2, Supplementary Fig. 2).
4. Discussion Intact, injured and necrotic cells can be seen in regions close to and distant from the hematoma in a rabbit model of ICH.17 When Qureshi et al. collected tissue specimens from boundary tissue after removing intracerebral hematomas in 14 patients, they found that 38% of cells in the perihematoma region had undergone apoptotic changes. However, in that study the number of patients was small.18 In the present study, we observed and assessed apoptosis on the basis of the morphological and staining characteristics of cells in 46 patients with ICH. Apoptosis was evident in 42 of the 46 surgical specimens available for analysis. In more than 10 patients, more than half of all cells were apoptotic. Interestingly, four patients had no apoptotic cells. There was no association between the percentage apoptotic cells and patient age or hematoma volume. The transcription factor NF-jB is a regulator of cell death and survival. Following experimental ICH in rats, Hickenbottom et al. found a 1.8–2.5-fold increase in NF-jB levels in ipsilateral perihematoma samples obtained 4 days after ICH.19 In the present study, activation of NF-jB varied with time after ICH. The number of immunostained neurons reached a maximum at 7–48 hours after ICH (p < 0.01, compared with control group). Furthermore, an increase in the number of immunoreactive neurons was associated with the degree of apoptosis. These findings suggest a relationship between NF-jB and the pathobiological mechanism of apoptotic cell death after ICH. Activation of NF-jB leads to rapid and coordinated induction of proteolytic enzymes and release of proinflammatory cytokines and chemokines. To further understand the potential implications of NF-jB activation, it is imperative to characterize the expression of proinflammatory cytokine and chemokine gene product changes after ICH. The proinflammatory contribution of IL-1b in various brain injury models, in neurodegeneration and in increased blood–brain-barrier permeability and edema has been well described.20–22 We found that the number of IL-1b-immunostained neurons reached a maximum at 2–6 hours after ICH (p < 0.01, compared with control group; Table 2). This means that expression of IL-1b may be regulated by the activation of NF-jB. ICAM-1 is an endothelial- and leukocyte-associated transmembrane protein long known for its importance in stabilizing cell–cell interactions and facilitating leukocyte endothelial transmigration. Because of these associations with immune responses, many researchers have hypothesized that ICAM-1 could function in signal transduction of immune reactions. In the present study, we showed that the number of ICAM-1-immunostained neurons was higher relative to control at 2–48 hours after ICH and reached a maximum at 49– 72 hours after ICH (p < 0.01 compared with control group). We hypothesize that ICAM-1 might act as a mediator of NF-jB proapoptotic activity after ICH. In the present study, we found that apoptosis plays an important role in cell death after ICH. That activation of NF-jB was positively associated with the progress of apoptosis indicates that NF-jB could be a vital mediator of apoptosis transduction. Table 2 Expression of nuclear factor-jB (NF-jB), interleukin-1b (IL-1b), and intercellular adhesion molecule-1 (ICAM-1) in sections of temporal gyrus from patients with hypertensive intracerebral hemorrhage (ICH) (n = 46) and controls (n = 5) Time since ICH (hours)
n
NF-jB
IL-1b
ICAM-1
2–6 7–48 49–72 Control
15 18 13 5
12.5 ± 3.9⁄ 30 ± 2.3⁄⁄ 24 ± 4.6⁄⁄ 6.1 ± 1.2
25.4 ± 7.2⁄⁄ 12.1 ± 3.6⁄ 13.0 ± 0.3⁄ 8.8.0 ± 1.1
2.3 ± 0.2⁄⁄ 10.7 ± 2.4⁄⁄ 17.6 ± 1.6⁄⁄ 0.6 ± 0.1
Data are mean ± standard deviation. ⁄p < 0.05 compared with control group, p < 0.01 compared with control group.
⁄⁄
Y.-X. Wang et al. / Journal of Clinical Neuroscience 18 (2011) 1392–1395
Fig. 2. Correlation between percentage of apoptotic cells and percentage nuclear factor-jB (NF-jB)-positive cells (%) in the middle temporal gyrus of 42 patients with intracerebral hemorrhage. There was a positive linear relationship between the two variables.
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