Nerve growth factor precursor and sortilin effects on perihematomal brain tissue and the relationship to secondary cell apoptosis*☆

Shiwen Guo, Yuliang Han, Gang Bao, Wenzhi Li

Department of Neurosurgery, First Affiliated Hospital, Medical College of Xi’an Jiaotong University, Xi’an  710061, Shaanxi Province, China

Shiwen Guo☆, Doctor, Chief physician, Professor, Doctoral supervisor, Department of Neurosurgery, First Affiliated Hospital,  Medical College of Xi’an Jiaotong University, Xi’an 710061, Shaanxi Province, China

Corresponding author: Yuliang Han, Master, Department of Neurosurgery, First Affiliated Hospital, Medical College of Xi’an Jiaotong University, Xi’an  710061, Shaanxi Province, China   
hyl_do@163.com

Supported by: Scientific and Technological Research Developing Program of Shaanxi Province, No. 2007K15-01*

Abstract
BACKGROUND: Neuronal apoptosis in perihematomal brain tissues following intracerebral hem-orrhage is strongly related to the formation of a compound signal pathway between nerve growth factor precursor (proNGF), p75NTR, and sortilin receptor. Sortilin acts as a co-receptor and mo-lecular switch governing the p75NTR-mediated pro-apoptotic signal induced by proNGF.
OBJECTIVE: To investigate proNGF and sortilin expressions in perihematomal brain tissues fol-lowing intracerebral hemorrhage, and to study the effects of proNGF and sortilin on secondary cell apoptosis.
DESIGN, TIME AND SETTING: A paired, comparison study was performed at the Laboratory of Histology and Embryology, Xi’an Jiaotong University from October 2007 to September 2008.
MATERIALS: Brain tissue samples were obtained from 15 patients with intracerebral hemorrhage, who were treated at the Department of Neurosurgery, First Affiliated Hospital, Medical College of Xi’an Jiaotong University from October 2007 to March 2008. Rabbit anti-proNGF polyclonal antibody was provided by Chemicon, USA; rabbit anti-sortilin polyclonal antibody by Abcam, UK; and TUNEL kit by Promega, USA.
METHODS: Perihematomal brain tissues selected 0.5 cm from the hemorrhage area were consid-ered to be the hemorrhage group, while brain tissues from the middle temporal gyrus served as the control group.
MAIN OUTCOME MEASURES: Histopathological changes were detected using hematoxylin-eosin staining, cell apoptosis was determined using the TUNEL method, and proNGF and sortilin ex-pressions were determined using immunohistochemistry.
RESULTS: Edema was clearly observed in perihematomal brain tissues, and infiltration of inflam-matory cells was visible, with the presence of irregular and necrotic bodies. The apoptotic rate in the hemorrhage group was significantly greater than in the control group (P < 0.01). Moreover, sortilin expression significantly increased (P < 0.01), but proNGF expression remained unchanged (P > 0.05). Correlation analysis suggested that sortilin expression positively correlated with apoptosis (rs = 0.648, P < 0.01).
CONCLUSION: proNGF expression was stable, but sortilin expression increased in perihematomal brain tissues following intracerebral hemorrhage, suggesting that sortilin acted as a co-receptor and molecular switch to govern p75NTR-mediated cell apoptosis.
Key Words: intracranial hemorrhage; cell apoptosis; nerve growth factor precursor; sortilin

INTRODUCTION
  
Neuronal apoptosis of perihematomal brain tissues following intracerebral hemorrhage is one of the major mechanisms for occurrence of secondary intracerebral hemorrhage[1-3]. Therefore, studies focused on the apoptotic pathway are of significance for the treatment of intracerebral hemorrhage. In particular, nerve growth factor precursor (proNGF)-induced p75NTR-mediated expression is one of methods for promoting cell apoptosis. proNGF mediates cellular apoptosis via the formation of a ligand-receptor compound with p75NTR and sortilin[4-7]. Additionally, sortilin acts as assistant receptor and molecular switch for controlling proNGF-induced p75NTR-mediated signals[8].
p75NTR-mediated apoptosis has been shown in amyotrophic lateral sclerosis[9], Alzheimer’s disease[10-12], spinal cord injury[13], cerebral ischemia[14], subacute spongiform encephalopathy[15], and age-dependent neurodegeneration[16]. Further studies are needed to determine whether the p75NTR-mediated apoptotic pathway is activated and proNGF and sortilin expressions are altered by intracerebral hemorrhage.
Hematoxylin-eosin staining and TUNEL were used to observe morphological changes in perihematomal brain tissues and cell apoptosis, respectively, following
intracerebral hemorrhage. Immunohistochemistry was used to determine proNGF and sortilin expressions to analyze the relationship with cellular apoptosis.

MATERIALS AND METHODS

Design
A paired, comparison study.
Time and setting
This study was performed at the Laboratory of Histology and Embryology, Xi’an Jiaotong University from October 2007 to September 2008.
Materials
A total of 15 patients with intracerebral hemorrhage (hemorrhage volume: supratentorial hemorrhage > 30 mL, cerebellar hemorrhage > 10 mL) were enrolled from the Department of Neurosurgery, First Affiliated Hospital, Medical College of Xi’an Jiaotong University from October 2007 to March 2008.
The general conditions are as follows:
        
        
        

Brain tissue samples were obtained following informed consent, and the study was approved by the local research ethics committee.
Methods
Reagents and equipment

Grouping and sample preparation
Brain tissue samples were prepared according to a previously described method of bone-flap craniotomy or key-hole surgery[17-18]. Perihematomal brain tissues were selected 0.5 cm from the hemorrhage area and were considered to be the hemorrhage group, while brain tissues from the middle temporal gyrus served as the control group. All brain tissue samples were fixed with formaldehyde, paraffin-embedded, and sectioned into 7-μm sections for hematoxylin-eosin, TUNEL, and immunohistochemical staining.
Hematoxylin-eosin staining
The sections were dewaxed in dimethyl benzene and stained with hematoxylin for 10 minutes, color-separated in 1% hydrochloric acid-alcohol mixture for 3 minutes, counterstained with eosin for three minutes, cleared with dimethyl benzene, and gum-mounted[19]. Brain tissue morphology was observed under an optical microscope.
Cellular apoptosis as determined by TUNEL
The sections were incubated in paraformaldehyde (4%) at room temperature for 15 minutes, incubated in 100 μL TdT at 37 °C for 1 hour, rinsed with 2 × SSC at room temperature for 15 minutes, and incubated in PBS containing 0.3% hydrogen peroxide and then incubated in 100 μL HRP-labeled streptavidin (1: 500) for 30 minutes. Thereafter, the sections were counterstained with DAB, dehydrated, dried, and mounted[20]. Cellular apoptosis was detected under an optical microscope.
proNGF and sortilin expression as determined by immunohistochemistry
The sections were dewaxed, hydrated, and incubated in 3% hydrogen peroxide to remove endogenous peroxidase. Antigen retrieval was performed by heating, and the sections were then blocked with goat serum for 20 minutes and subsequently stained according to previously described methods[21]. The sections were incubated in rabbit anti-sortilin polyclonal antibody (1: 700) and rabbit anti-proNGF polyclonal antibody (1: 250) at  4 °C overnight, with biotin-labeled goat anti-rabbit IgG at 37 °C for 20 minutes, and alkaline phosphatase-labeled streptavidin at 37 °C for 30 minutes. The sections were then stained with DAB, counterstained with hematoxylin, dried, and mounted[21]. proNGF and sortilin expressions were detected under an optical microscope.
Quantification of TUNEL and immunohistochemistry-positive neurons
Three visual fields were randomly selected from each section using 400 × magnification to quantify the total number of cells and positively stained cells. The positive cell ratio was then calculated, i.e., positive cell ratio = (number of positive cells/total number of cells) × 100%. The mean value from three visual fields was considered to be the final positive cell rate.
Main outcome measures
Histopathological changes, cell apoptosis, and proNGF and sortilin expressions of the brain tissues.
Statistical analysis
SPSS 13.0 software (SPSS, Chicago, IL, USA) was used for statistical analysis. Data were expressed as Mean ± SD. TUNEL-positive, proNGF-positive, and sortilin-positive cell ratios were compared using the paired (1: 1) t-test between the hemorrhage and control groups. TUNEL-positive and sortilin-positive ratios were analyzed using the Spearman correlation analysis. P values of < 0.05 were considered statistically significant.

RESULTS

Morphological changes in brain tissues
Hematoxylin-eosin staining revealed noticeable edema in perihematomal brain tissues. Neurons and glial cells exhibited a large volume, with various sized vacuoles. Some neuronal membranes were broken, cytoplasm was lightly stained, and the nucleus was atrophied and darkly stained, with the presence of irregular and necrotic bodies. Blood capillaries were enlarged and engorged, and the vessel walls were swollen, with the presence of neutrophilic granulocyte infiltration and erythrocytes (Figure 1).

In the control group, edema was mild, neurons and glial cells were not swollen, occasional necrotic neurons were evident, the nucleus was not atrophied or darkly stained, the matrix was densely distributed around the cells, and the space was not obviously enlarged.
Cellular apoptosis
TUNEL staining revealed a large number of glial cells with brown-stained nucleus in the perihematomal brain tissues, i.e., apoptotic cells (Figure 2).

Several glial cells with brown-stained nucleus were observed in the control group, and the positive cell ratio was low. In addition, there were fewer neurons, and the positive cells accounted for 1/3 of the total number.
The number of apoptotic cells revealed that the apoptotic rate significantly increased in the hemorrhage group compared with the control group (P < 0.01; Table 1).

proNGF and sortilin expressions
Immunohistochemical staining revealed proNGF and sortilin expression in the hemorrhage and control groups (Figures 3, 4).

Statistical analysis demonstrated that the sortilin-positive cell ratio significantly increased in the hemorrhage group compared with the control group (P < 0.01), but there was no significant difference in the proNGF-positive cell ratio between the hemorrhage and control groups (P > 0.05; Table 1).

Correlation between sortilin and apoptotic cells in perihematomal brain tissues
The Spearman correlation analysis suggested that the sortilin-positive cell ratio positively correlated with the TUNEL-positive cell ratio (rs = 0.65, P < 0.01) (Figure 5).

DISCUSSION

Studies have shown that cellular apoptosis takes place in perihematomal brain tissues following intracerebral hemorrhage, which possibly correlates with decreased local cerebral blood flow and energy metabolism[22-23], inflammatory reactions in perihematomal brain tissues, and release of thrombin[1]. Results from the present study suggested that apoptotic cells, in particular neurons and glial cells, were observed in all perihematomal brain tissue samples. Additionally, TUNEL-positive cells were observed in the control group, suggesting that distance from sample selection to hemorrhagic focuses might induce various effects on intracerebral hemorrhage. The paired t-test analysis revealed that the apoptotic ratio significantly increased in the hemorrhage group compared with the control group (P < 0.01), suggesting that cellular apoptosis was an important factor contributing to secondary cerebral injury following intracerebral hemorrhage.
Previous reports have demonstrated that NGF increases neuronal survival, and is considered to be a polypeptide growth factor that promotes neuronal survival and growth. However, proNGF does not exhibit biological activity[24-25]. According to a previous study[26], proNGF induces cellular apoptosis. High-affinity proNGF combined with the target cell membrane p75NTR receptor induces p75NTR-mediated cell apoptosis. Moreover, high-affinity mNGF combined with the Trk receptor increases neuronal survival and axonal growth[4-7].
Results from the present study demonstrated no significant difference in proNGF protein expression between the hemorrhage and control groups (P > 0.05). However, increased proNGF expression has been shown in a trauma model and other rat models of disease[14]. proNGF protein expression was not altered in this study, suggesting that proNGF did not exhibit any effects on cellular apoptosis following intracerebral hemorrhage. Previous observations have demonstrated that proNGF expression in the hemorrhage group enhances p75NTR expression, as well as the number of apoptotic cells, indicating that proNGF-induced cellular apoptosis or cell survival correlates with cleavage of proNGF[27]. A previous study[28] has shown that proNGF cleavage induces decreased NGF expression in a rat model of retinal degeneration. Therefore, proNGF cleavage could be altered following intracerebral hemorrhage. Furthermore, cellular apoptosis following intracerebral hemorrhage promoted binding between proNGF and p75NTR, due to increased p75NTR expression at the cell membrane surface. However, combined proNGF resulting in internalization was not detected by immunohistochemistry. proNGF cleavage likely plays a key role in regulating and controlling biological actions of neurotrophic factors and secondary cerebral injury.
Sortilin is considered to be a necessary factor for proNGF induction of p75NTR-mediated cell death. Sortilin-specific antibodies have been shown to block cell death[29], and sortilin knockout mice were similar to p75NTR knockout mice, exhibiting decreased apoptosis[21, 30-31]. Sortilin, p75NTR, and proNGF form a trimer to induce biological action[32]. Sortilin acts as an assistant receptor and molecular switch to control signals prior to p75NTR-mediated proNGF-induced  apoptosis[29, 33].
In the present study, sortilin protein expression increased in perihematomal brain tissues, and correlation analysis revealed that sortilin expression positively correlated with the number of apoptotic cells. Although proNGF expression was not altered following intracerebral hemorrhage, sortilin expression increased and positively correlated with the number of apoptotic cells. Sortilin has been shown to control the transformation from proNGF to mNGF[29]. When sortilin expression increased, proNGF expression increased, the proNGF-sortilin-p75NTR trimer increased, and the neurotrophic effect of mNGF decreased. In contrast, increased sortilin expression promoted the combination with proNGF, but did not influence proNGF expression.
In conclusion, proNGF expression was not altered in perihematomal brain tissues following intracerebral hemorrhage. Sortilin expression increased, and positively correlated with cellular apoptosis. Sortilin acted as an assistant receptor and molecular switch for p75NTR-mediated apoptosis. It is important to note that samples in this study were randomly collected from 5 hours to 4 days following intracerebral hemorrhage. Therefore, the analyses of cellular apoptosis, and proNGF and sortilin expressions were not systematic, and the sample numbers were limited.

REFERENCES

[1] Qureshi AI, Suri MF, Ostrow PT, et al. Apoptosis as a form of cell death in intracerebral hemorrhage. Neurosurgery. 2003;52(5): 1041-1047.
[2] 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(1):152-157.
[3] Matsushita K, Meng W, Wang X, et al. Evidence for apoptosis after intercerebral hemorrhage in rat striatum. J Cereb Blood Flow Metab. 2000;20(2):396-404.
[4] Hempstead BL. Dissecting the diverse actions of pro- and mature neurotrophins. Curr Alzheimer Res. 2006;3(1):19-24.
[5] Skaper SD. The biology of neurotrophins, signalling pathways, and functional peptide mimetics of neurotrophins and their receptors. CNS Neurol Disord Drug Targets. 2008;7(1):46-62.
[6] Paiardini A, Caputo V. Insights into the interaction of sortilin with proneurotrophins: a computational approach. Neuropeptides. 2008;42(2):205-214.
[7] Clewes O, Fahey MS, Tyler SJ, et al. Human ProNGF: biological effects and binding profiles at TrkA, P75NTR and sortilin. J Neurochem. 2008;107(4):1124-1135.
[8] Willnow TE, Petersen CM, Nykjaer A. VPS10P-domain receptors - regulators of neuronal viability and function. Nat Rev Neurosci. 2008;9(12):899-909.
[9] Turner BJ, Cheah IK, Macfarlane KJ, et al. Antisense peptide nucleic acid-mediated knockdown of the p75 neurotrophin receptor delays motor neuron disease in mutant SOD1 transgenic mice. J Neurochem. 2003;87(3):752-763.
[10] Hashimoto Y, Kaneko Y, Tsukamoto E, et al. Molecular characterization of neurohybrid cell death induced by Alzheimer's amyloid-beta peptides via p75NTR/PLAIDD. J Neurochem. 2004;90(3):549-558.
[11] Peng S, Wuu J, Mufson EJ, et al. Increased proNGF levels in subjects with mild cognitive impairment and mild Alzheimer disease. J Neuropathol Exp Neurol. 2004;63(6):641-649.
[12] Cuello AC, Bruno MA. The failure in NGF maturation and its increased degradation as the probable cause for the vulnerability of cholinergic neurons in Alzheimer's disease. Neurochem Res. 2007;32(6):1041-1045.
[13] Beattie MS, Harrington AW, Lee R, et al. ProNGF induces p75-mediated death of oligodendrocytes following spinal cord injury. Neuron. 2002;36(3):375-386.
[14] Harrington AW, Leiner B, Blechschmitt C, et al. Secreted proNGF is a pathophysiological death-inducing ligand after adult CNS injury. Proc Natl Acad Sci U S A. 2004;101(16):6226-6230.
[15] Stoica G, Lungu G, Kim HT, et al. Up-regulation of pro-nerve growth factor, neurotrophin receptor p75, and sortilin is associated with retrovirus-induced spongiform encephalomyelopathy. Brain Res. 2008;1208:204-216.
[16] Al-Shawi R, Hafner A, Chun S, et al. ProNGF, sortilin, and age-related neurodegeneration. Ann N Y Acad Sci. 2007;1119: 208-215.
[17] Thiex R, Tsirka SE. Brain edema after intracerebral hemorrhage: mechanisms, treatment options, management strategies, and operative indications. Neurosurg Focus. 2007;22(5):E6.
[18] Nakano T, Ohkuma H, Ebina K, et al. Neuroendoscopic surgery for intracerebral haemorrhage--comparison with traditional therapies. Minim Invasive Neurosurg. 2003;46(5):278-283.
[19] Hua Y, Nakamura T, Keep RF, et al. Long-term effects of experimental intracerebral hemorrhage: the role of iron. J Neurosurg. 2006;104(2):305-312.
[20] Han N, Ding SJ, Wu T, et al. Correlation of free radical level and apoptosis after intracerebral hemorrhage in rats. Neurosci Bull. 2008;24(6):351-358.
[21] Jansen P, Giehl K, Nyengaard JR, et al. Roles for the pro-neurotrophin receptor sortilin in neuronal development, aging and brain injury. Nat Neurosci. 2007;10(11):1449-1457.
[22] Adams RE, Diringer MN. Response to external ventricular drainage in spontaneous intracerebral hemorrhage with hydrocephalus. Neurology. 1998;50(2):519-523.
[23] Mendelow AD. Mechanisms of ischemic brain damage with intracerebral hemorrhage. Stroke. 1993;24(12 Suppl):I115-117.
[24] Mitsiadis TA, Couble P, Dicou E, et al. Patterns of nerve growth factor (NGF), proNGF, and p75 NGF receptor expression in the rat incisor: comparison with expression in the molar. Differentiation. 1993;54(3):161-175.
[25] Seidah NG, Benjannet S, Pareek S, et al. Cellular processing of the nerve growth factor precursor by the mammalian pro-protein convertases. Biochem J. 1996;314(Pt 3):951-960.
[26] Lee R, Kermani P, Teng KK, et al. Regulation of cell survival by secreted proneurotrophins. Science. 2001;294(5548):1945-1948.
[27] Fahnestock M, Yu G, Michalski B, et al. The nerve growth factor precursor proNGF exhibits neurotrophic activity but is less active than mature nerve growth factor. J Neurochem. 2004;89(3): 581-592.
[28] Srinivasan B, Roque CH, Hempstead BL, et al. Microglia-derived pronerve growth factor promotes photoreceptor cell death via p75 neurotrophin receptor. J Biol Chem. 2004;279(40):41839-41845.
[29] Nykjaer A, Lee R, Teng KK, et al. Sortilin is essential for proNGF-induced neuronal cell death. Nature. 2004;427(6977): 843-848.
[30] Provenzano MJ, Xu N, Ver Meer MR, et al. p75NTR and sortilin increase after facial nerve injury. Laryngoscope. 2008;118(1): 87-93.
[31] Al-Shawi R, Hafner A, Olsen J, et al. Neurotoxic and neurotrophic roles of proNGF and the receptor sortilin in the adult and ageing nervous system. Eur J Neurosci. 2008;27(8):2103-2114.
[32] Bl?chl A, Bl?chl R. A cell-biological model of p75NTR signaling. J Neurochem. 2007;102(2):289-305.
[33] Nakamura K, Namekata K, Harada C, et al. Intracellular sortilin expression pattern regulates proNGF-induced naturally occurring cell death during development. Cell Death Differ. 2007;14(8): 1552-1554.
 (Edited by Li ZX, Yu MK, Yao CY/Ji H/Song LP)