人脑挫裂伤早期水通道蛋白4及血脑屏障的变化

doi:10.3969/j.issn.1673-5374.2013.04.006

摘要/Abstract

摘要：

实验对人脑挫裂伤后2~72h损伤组织水通道蛋白4表达和血脑屏障超微结构的改变进行研究，探讨其与脑水肿形成的关系。结果发现，脑挫裂伤后2 h，水通道蛋白4表达明显增加，同时脑含水量和血脑屏障通透性增加，脑微血管内皮细胞吞饮小泡增多，线粒体堆积。随着脑挫裂伤时间的延长，水通道蛋白4表达持续增多，脑含水量和血脑屏障通透性也逐渐增加，脑组织毛细血管内皮细胞、星形胶质细胞肿胀及毛细血管基底膜损伤也逐渐加重，至伤后12h达高峰，之后逐渐下降。且水通道蛋白4表达与脑含水量及血脑屏障指数均呈显著正相关。提示人脑挫裂伤后水通道蛋白4的表达及血脑屏障通透性的增加参与了脑水肿的形成。

关键词: 神经再生, 脑损伤, 脑挫裂伤, 水通道蛋白4, 血脑屏障, 超微结构, 脑水肿, 人, 早期, 图片文章

Abstract:

This study aimed to investigate aquaporin 4 expression and the ultrastructure of the blood-brain barrier at 2–72 hours following cerebral contusion injury, and correlate these changes to the formation of brain edema. Results revealed that at 2 hours after cerebral contusion and laceration injury, aquaporin 4 expression significantly increased, brain water content and blood-brain barrier permeability increased, and the number of pinocytotic vesicles in cerebral microvascular endothelial cells increased. In addition, the mitochondrial accumulation was observed. As contusion and laceration injury became aggravated, aquaporin 4 expression continued to increase, brain water content and blood-brain barrier permeability gradually increased, brain capillary endothelial cells and astrocytes swelled, and capillary basement membrane injury gradually increased. The above changes were most apparent at 12 hours after injury, after which they gradually attenuated. Aquaporin 4 expression positively correlated with brain water content and the blood-brain barrier index. Our experimental findings indicate that increasing aquaporin 4 expression and blood-brain barrier permeability after cerebral contusion and laceration injury in humans is involved in the formation of brain edema.

Key words: neural regeneration, brain injury, cerebral contusion and laceration injury, aquaporin 4, blood-brain barrier, ultrastructure, brain edema, human, early stage, photographs-containing paper, neuroregeneration

. 人脑挫裂伤早期水通道蛋白4及血脑屏障的变化[J]. 中国神经再生研究(英文版), 2013, 8(4): 338-345.

Xinjun Li, Yangyun Han, Hong Xu, Zhongshu Sun, Zengjun Zhou, Xiaodong Long. Aquaporin 4 expression and ultrastructure of the blood-brain barrier following cerebral contusion injury[J]. Neural Regeneration Research, 2013, 8(4): 338-345.

参考文献

[1] Bloch O, Manley GT. The role of aquaporin-4 in cerebral water transport and edema. Neurosurg Focus. 2007;22(5):E3.

[2] Papadopoulos MC, Verkman AS. Aquaporin-4 and brain edema. Pediatr Nephrol. 2007;22(6):778-784.

[3] Amiry-Moghaddam M, Hoddevik EH, Ottersen OP. Aquaporins: multifarious roles in brain. Neuroscience. 2010;168(4):859-861.

[4] Xu M, Su W, Xu QP. Aquaporin-4 and traumatic brain edema. Chin J Traumatol. 2010;13(2):103-110.

[5] Iacovetta C, Rudloff E, Kirby R. The role of aquaporin 4 in the brain. Vet Clin Pathol. 2012;41(1):32-44.

[6] Nico B, Ribatti D. Morphofunctional aspects of the blood-brain barrier. Curr Drug Metab. 2012;13(1):50-60.

[7] Liebner S, Czupalla CJ, Wolburg H. Current concepts of blood-brain barrier development. Int J Dev Biol. 2011; 55(4-5):467-476.

[8] Zhang L, Yuan F. Enhanced expression of aquaporin-4 aggravates brain edema after traumatic brain injury in rat. Shoudu Yike Daxue Xuebao. 2008;29(6):732-736.

[9] Shen LQ, Jiang JQ, Guo JJ. Expression and significance of AQP4 in contusion brain tissue. Shandong Yiyao. 2011; 26(1):6-7.

[10] Taheri S, Gasparovic C, Huisa BN, et al. Blood-brain barrier permeability abnormalities in vascular cognitive impairment. Stroke. 2011;42(8):2158-2163.

[11] Venero JL, Vimete ML, Machado A, et al. Aquaporins in the central nervous system. Prog Neumbiol. 2001;63(3): 321-336.

[12] Saadoun S, Papadopoulos MC. Aquaporin-4 in brain and spinal cord oedema. Neuroscience. 2010;168(4):1036-1046.

[13] Zador Z, Stiver S, Wang V, et al. Role of aquaporin-4 in cerebral edema and stroke. Handb Exp Pharmacol. 2009;(190):159-170.

[14] Arciénega II, Brunet JF, Bloch J, et al. Cell locations for AQP1, AQP4 and 9 in the non-human primate brain. Neuroscience. 2010;167(4):1103-1114.

[15] Masato Y. Regulation, structure and function of brain aquaporin. Rinsho Shinkeigaku. 2009;49(11):786-788.

[16] Fenton RA, Moeller HB, Zelenina M, et al. Differential water permeability and regulation of three aquaporin 4 isoforms. Cell Mol Life Sci. 2010;67(5):829-840.

[17] Yukutake Y, Yasui M. Regulation of water permeability through aquaporin-4. Neuroscience. 2010;168(4): 885-891.

[18] Romeiro RR, Romano-Silva MA, De Marco L, et al. Can variation in aquaporin 4 gene be associated with different outcomes in traumatic brain edema? Neurosci Lett. 2007;426(2):133-134.

[19] Sun MC, Honey CR, Berk C, et al. Regulation of aquaporin-4 in a traumatic brain injury model in rats. J Neurosurg. 2003;98(3):565-569.

[20] Taya K, Marmarou CR, Okunok K, et al. Effect of secondary insults upon aquaporin-4 water channels following experimental cortical contusion in rats. J Neurotrauma. 2010;27(1):229-239.

[21] Bonomini F, Rezzani R. Aquaporin and blood brain barrier. Curr Neuropharmacol. 2010;8(2):92-96.

[22] Saadoun S, Tait MJ, Reza A, et al. AQP4 gene deletion in mice does not alter blood-brain barrier integrity or brain morphology. Neuroscience. 2009;161(3):764-772.

[23] Zhou J, Kong H, Hua X, et al. Altered blood-brain barrier integrity in adult aquaporin-4 knockout mice. Neuroreport. 2008;19(1):1-5.

[24] Wolburg H, Noell S, Wolburg-Buchholz K, et al. Agrin, aquaporin-4, and astrocyte polarity as an important feature of the blood-brain barrier. J Neuroscientist. 2009; 15(2):180-193.

[25] Higashida T, Kreipke CW, Rafols JA, et al. The role of hypoxia-inducible factor-1α, aquaporin-4, and matrix metalloproteinase-9 in blood-brain barrier disruption and brain edema after traumatic brain injury. J Neurosurg. 2011;114(1):92-101.

[26] Shi WZ, Zhao CZ, Huang XQ, et al. Deficiency of water channel AQP4 aggravates NMDA-induced brain injury in mice. Zhejiang Daxue Xuebao: Yixue Ban. 2011;40(2): 145-149.

[27] Ke C, Poon WS, NG HK, et al. Heterogeneous responses of aquaporin-4 in edema formation in a replicated severe traumatic brain injury model in rats. Neurosci Lett. 2001; 301(1):21-24.

[28] Zhu SM, Xiong XX, Zheng YY, et al. Propofol inhibits aquaporin 4 expression through a protein kinase C- dependent pathway in an astrocyte model of cerebral ischemia/reoxygenation. Anesth Analg. 2009;109(5): 1493-1499.

[29] Karasu A, Aras Y, Sabanc? PA, et al. The effects of protein kinase C activator phorbol dibutyrate on traumatic brain edema and aquaporin-4 expression. Ulus Travma Acil Cerrahi Derg. 2010;16(5):390-394.

[30] Wang P, Ni RY, Chen MN, et al. Expression of aquaporin-4 in human supratentorial meningiomas with peritumoral brain edema and correlation of VEGF with edema formation. Genet Mol Res. 2011;10(3):2165-2171.

[31] Rao KV, Reddy PV, Curtis KM, et al. Aquaporin-4 expression in cultured astrocytes after fluid percussion injury. J Neurotrauma. 2011;28(3):371-381.

[32] Thrane AS, Rappold PM, Fujita T, et al. Critical role of aquaporin-4 (AQP4) in astrocytic Ca2+ signaling events elicited by cerebral edema. Proc Natl Acad Sci U S A. 2011;108(2):846-851.

[33] Katada R, Nishitani Y, Honmou O, et al. Expression of aquaporin-4 augments cytotoxic brain edema after traumatic brain injury during acute ethanol exposure. Am J Pathol. 2012;180(1):17-23.

[34] Tanimura Y, Hiroaki Y, Fujiyoshi Y. Acetazolamide reversibly inhibits water conduction by aquaporin-4. J Struct Biol. 2009;166(1):16-21.

[35] Igarashi H, Huber VJ, Tsujita M, et al. Pretreatment with a novel aquaporin 4 inhibitor, TGN-020, significantly reduces ischemic cerebral edema. Neurol Sci. 2011;32(1): 113-116.

[36] Zeng HK, Wang QS, Deng YY, et al. Hypertonic saline ameliorates cerebral edema through downregulation of aquaporin-4 expression in the astrocytes. Neuroscience. 2010;166(3):878-885.

[37] Tang Y, Cai D, Chen Y. Thrombin inhibits aquaporin 4 expression through protein kinase C-dependent pathway in cultured astrocytes. J Mol Neurosci. 2007;31(1):83-93.

[38] Fazzina G, Amorini AM, Marmarou CR, et al. The protein kinase C activator phorbol myristate acetate decreases brain edema by aquaporin 4 downregulation after middle cerebral artery occlusion in the rat. J Neurotrauma. 2010;27(2):453-461.

[39] Belayev L, Busto R Zhao W, et al. Quantitative evaluation of blood-brain barrier permeability following middle cerebral artery occlusion in rats. Brain Res. 1996;739(1-2):88-96.

[40] Wang YJ. Neurology Laboratory Diagnostic Techniques. Beijing: Science and Technology Literature Publishing House. 1998.

引言

材料方法

Design

An open randomized controlled experiment.

Time and setting

Experiments were performed in the Molecular Biology Laboratory, Deyang People’s Hospital and the Neural Biological Laboratory of the Third Military Medical University of Chinese PLA from February 2011 to January 2012.

Materials

Brain tissue specimens

Seventy patients with cerebral contusion and laceration injury, confirmed by craniocerebral CT, and with surgical indications (CT confirmed the midline shifted greater than 1 cm; supplementary Figure 1 online), aged 30–40 years, were involved in this study and agreed to receive surgery. Patients with great variations in age, operation mode and disease conditions were excluded. Among the patients, 40 were men and 30 were women, with a mean age of 31.34 ± 4.25 years. The site of injury included the frontal lobe in 52 cases, temporal lobe in 8 cases, and cerebellum in 10 cases. Cerebral contusion and laceration group: brain tissue specimens harvested from a 3-cm area in the center of the injury area in 60 patients; control group: non-functional normal brain tissue from 10 patients (frontal or temporal pole brain tissue resected due to internal decompression during operation).

The cerebral contusion and laceration group was further assigned into six subgroups according to the operation time, namely 2, 6, 8, 12, 24, and 72 hours.

Cerebrospinal fluid and venous blood sampling

Cerebrospinal fluid was surgically collected through puncture or lumbar puncture for the determination of cerebrospinal fluid albumin. Elbow vein blood was extracted for serum albumin detection.

All involved patients and their relatives agreed to the experiment and signed the informed consent. The experimental disposals complied with the Helsinki Declaration ethical standard.

Methods

Brain water content determination

Brain tissue 150 mg were weighed for wet weight, placed in a 100°C constant temperature box for 24 hours, and then weighed for dry weight. Brain water content was calculated according to the following formula: (wet weight – dry weight)/wet weight × 100%, to represent the degree of brain edema^[39].

Immunohistochemical detection of aquaporin 4 expression

Brain tissue specimens at 1 mm thick were fixed with 4% (w/v) paraformaldehyde for 6 hours and rinsed for 12 hours, followed by gradient ethanol dehydration, chloroform, ethanol xylene and waxing, and soft paraffin embedding into blocks. Specimens were cut into slices using an automatic microtome, at 5 µm thickness. After conventional dewaxing, slices were incubated with 3% (v/v) H₂O₂ for 10 minutes at room temperature, blocked with sheep serum for 20 minutes, and incubated with rabbit anti-aquaporin 4 polyclonal antibody (1:50; Abnova, Walnut, CA, USA) at 4°C overnight. Sections were then incubated with biotinylated-conjugated goat anti-rabbit IgG (1:100; Abnova) for 1 hour at room temperature, and with SABC (Abnova) at 37°C for 20 minutes. Diaminobenzidine (Sigma, St. Louis, MO, USA) was used for coloration, followed by routine dehydration, transparency and mounting. Finally, slices were observed under a light microscope (Olympus, Tokyo, Japan). Positive staining was observed as yellow fine particles in the cell membrane. Using the Image-Pro-Plus image analysis system (Media Cybernetics, Inc. Bethesda, MD, USA), the staining of aquaporin 4 was semi-quantitatively analyzed using five randomly selected cerebral cortical slices from each specimen, and five cerebral cortical areas on each slice.

Blood-brain barrier index determination

Cerebrospinal fluid and serum were harvested to determine cerebrospinal fluid and serum albumin levels using a CX7 automatic biochemical analyzer (Beckman Coulter, Inc, Fullerton, CA, USA). According to the formula formulated by Wang^[40], the blood-brain barrier index was calculated as follows: blood-brain barrier index = cerebrospinal fluid albumin/serum albumin.

Blood-brain barrier ultrastructure

Normal brain tissue and brain tissue fixed at each time point (1.0 mm × 1.0 mm × 1.0 mm) were fixed in glutaraldehyde and 1% (w/v) osmium tetroxide, dehydrated in acetone, embedded with 618 epoxy resin in situ, and prepared into ultrathin sections (60 nm) for double lead staining. Sections were observed under a transmission electron microscope (JEM-2000; Sanyo, Tokyo, Japan).

讨论

文章快阅

延伸阅读

1 实验设计构思介绍：

观察人脑挫裂伤后水通道蛋白4和血脑屏障超微结构在脑水肿形成中的不同时间点的变化特征，探讨脑水肿的形成机制，为颅脑损伤后脑水肿的治疗和预后提供客观依据。

（1）选取经颅脑CT证实为脑挫裂伤且需手术治疗的患者。

（2）手术过程切取小部分正常脑组织及挫裂伤脑组织，随机分为对照组和脑挫裂伤组。对照组不做任何处理，脑挫裂伤组于按时间伤后2 h、6h、8h、12 h、24 h、72h分6个亚组。

（3）采用免疫组化和图像分析技术测定各组相应时间点水肿区水通道蛋白4的表达水平，同时用干湿重法检测脑水肿含水量，检测血脑屏障通透性;并应用光镜、透射电镜对相应的部位进行病理观察，观察血脑屏障超微结构的变化。

（4）对上述数据进行统计分析，得出结论，并针对不同时间点脑水肿的治疗提供理论依据。

2 国内外同类研究水平的介绍：

国内已有脑挫伤组织水通道蛋白-4的表达的动物实验研究文献报道，但关于人脑挫伤组织水通道蛋白-4的表达及血脑屏障超微结构变化的研究文献报道较少。

3与其他同类研究的比较：

人脑挫伤组织中水通道蛋白-4的表达与血脑屏障超微结构变化之间的相关性统计学分析研究，及与血脑屏障超微结构变化的相关性及变化趋势的分析研究，国内未查见有相同的研究文献报道。

（沈礼芹;蒋金泉;郭建杰等。脑挫裂伤后脑组织中水通道蛋白4的表达变化及意义。《山东医药》2011年第26期。采用免疫组化法检测20例标本中水通道蛋白4,同时观察血脑屏障(血脑屏障)指数及脑水肿的变化，未应用光镜、透射电镜对相应的部位进行病理观察，观察血脑屏障超微结构的变化。）

4 文章先进性：

（1）对象：国内外对脑出血后脑水肿形成机制等研究较多，部分学者对脑挫裂伤后脑水肿的形成机制的动物实验进行了基础研究，但对人颅脑损伤后脑挫裂伤后脑水肿的形成机制研究甚少。本课题在人脑组织进行基础性研究为临床治疗提供直接的依据。不同于以往动物实验。迄今我们尚未见文献报道。本课题所作的工作将填补这方面的空白。

（2）指标参数：脑挫裂伤周围组织水通道蛋白4表达及血脑屏障超微结构变化，为目前研究脑水肿最具有意义的观察指标。

（3）方法：采用免疫组化和图像分析技术测定各组相应时间点水肿区水通道蛋白4的表达水平；用干湿重法检测脑水肿含水量，检测血脑屏障（血脑屏障）通透性；应用光镜、透射电镜对相应的部位进行病理观察，观察血脑屏障超微结构的变化。是目前较先进及公认的技术。

（4）结果：实验结果来源于真正的颅脑损伤患者，数据直观、可靠，为颅脑损伤后脑水肿的治疗与预后提供了直接可靠的临床依据。

5查新报告：

四川省医学情报研究所（国家卫生部、四川省科技厅科技查新认定单位）：报告编号：2012036。委托查新课题申报的脑挫裂伤早期周围组织水通道蛋白4表达及血脑屏障超微结构变化研究，拟进行人脑组织脑挫裂伤后脑组织中水通道蛋白4的表达和观察血脑屏障超微结构的变化的研究。经与所查国内相关文献对比分析，得出以下查新结论，在检索文献范围内：

（1）国内已有脑挫伤组织水通道蛋白-4的表达的动物实验研究文献报道，但关于人脑挫伤组织水通道蛋白-4的表达及血脑屏障超微结构变化的研究文献报道较少。

（2）委托查新课题拟进行的人脑挫伤组织中水通道蛋白-4的表达与血脑屏障超微结构变化之间的相关性统计学分析研究，国内未查见有相同的研究文献报道。

委托查新课题拟进行的人脑挫伤组织中水通道蛋白4与脑水肿并与血脑屏障超微结构变化的相关性及变化趋势的分析研究，国内未查见有相同的研究文献报道。