Cerebroprotection with recombinant neuroglobin plasmid in a rat model of focal cerebral ischemia*☆

Ji Zhu, Wenyuan Tang

Department of Neurosurgery, First Affiliated Hospital of Chongqing Medical University, Chongqing  400016, China

Ji Zhu☆, Doctor, Associate professor, Associate chief physician, Department of Neurosurgery, First Affiliated Hospital of Chongqing Medical University, Chongqing  400016, China

Supported by: a Grant from Chongqing Health Bureau, No. 06-2-177*

Received: 2009-07-23
Accepted: 2009-11-20    (NY20090223006/H)

Zhu J, Tang WY. Cerebroprotection with recombinant neuroglobin plasmid in a rat model of focal cerebral ischemia. Neural Regen Res. 2010;5(1):52-57.

Abstract
BACKGROUND: Adenovirus has been used to develop neuroglobin (Ngb) vectors. Although transfection efficiency is high, induced gene mutation, cytotoxicity, inflammation, and low exogenous gene content have limited its application.
OBJECTIVE: To observe the effects of recombinant Ngb plasmid in a rat model of focal cerebral ischemia.
DESIGN, TIME AND SETTING: Genetically engineered, randomized, controlled, animal experiment was performed at the Laboratory of Chongqing Medical University from May 2006 and January 2007.
MATERIALS: 2, 3, 5-triphenyltetrazolium chloride was purchased from Shanghai Sangon Biological Engineering Technology and Services. Rabbit anti-rat Bcl-2 polyclonal antibody, rabbit anti-rat β-actin monoclonal antibody, and FITC-labeled goat anti-rabbit IgG were purchased from Sigma, USA. TUNEL apoptosis kit was purchased from Roche, Germany.
METHODS: A total of 54 male, adult, Wistar rats were randomly assigned to 3 groups (n = 18): normal saline, plasmid control, and recombinant Ngb (pCDNA3.1(+)/Ngb). Normal saline, plasmid pCDNA3.1(+), and recombinant plasmid pCDNA3.1(+)/Ngb were separately injected into two sites in the rat cerebral cortex, and models of focal ischemia were established by occlusion of the right middle cerebral artery after 24 hours.
MAIN OUTCOME MEASURES: Local ischemic damage was detected by 2, 3, 5- triphenyltetra-zolium chloride staining, apoptosis in the penumbra was confirmed using the TUNEL method, and Bcl-2 protein expression in the penumbra was determined by indirect immunofluorescent staining and Western blot analysis.
RESULTS: Compared with the normal saline and plasmid control groups, cerebral infarction size and the number of apoptotic cells in the pCDNA3.1(+)/Ngb group were significantly reduced (P < 0.01). The percentage of Bcl-2-positive cells in the penumbra of the pCDNA3.1(+)/Ngb group was significantly increased (P < 0.01). The relative expression level of Bcl-2 protein was increased by 40%–50%.
CONCLUSION: Recombinant plasmid pCDNA3.1/Ngb provides neuroprotection by upregulating Bcl-2 expression and inhibiting cell apoptosis in the penumbra.
Key Words: neuroglobin; cerebral ischemia; cell apoptosis; ischemic penumbra; Bcl-2

INTRODUCTION
  
Neuroglobin (Ngb) was first discovered by Burmester in 2000[1]. Similar to hemoglobin and myoglobin, Ngb is an oxygen-carrying globulin and is predominantly localized in brain tissues of vertebrates. It has been shown to participate in stress processes, such as brain cell ischemia and hypoxia[2-3]. Delayed neuronal injury following cerebral ischemia is always accompanied by apop-tosis, and Bcl-2 is considered an important endogenous anti-apoptotic factor[4] that plays an essential role in maintaining neu-ronal survival following focal cerebral ischemia/reperfusion[5]. Studies have shown that the high oxygen affinity of Ngb allows for reversible binding to oxygen. In addition, as an oxygen-carrier, Ngb enhances oxygen diffusion into mitochondria and neuronal oxygen metabolism[6]. Hypoxia-induced Ngb expression attenuates neuronal damage, and reduced Ngb expression aggravates neuronal damage in cultured cerebral corti-cal cells[7]. An in vitro study demonstrated that Ngb is an endogenous neuroprotective factor for pathological cerebral ischemia[8].
The ideal vector for gene therapy carries genes to specific cells or tissues and effec-tively controls gene expression, but does not induce an immunological or inflammatory reaction or potential gene mutation. The vectors currently in use for gene therapy do not meet these requirements. In particular, efficient gene transfection is difficult in gene therapy for the central nervous system[9]. Previous studies have utilized transgenic animal models or various viruses as vec-tors[10]. Although transfection efficiency is high, 
these systems are limited for clinical application due to induced gene mutation, cytotoxicity, induced inflammation, and limited exogenous gene content. However, studies have demonstrated that transgenic vector plasmid effec-tively transfer exogenous genes to neurons, leading to increased gene expression[11]. 
The present study established a rat model of cerebral ischemia. Using a plasmid as a vector, pCDNA3.1(+)/Ngb was constructed by genetic engineering and introduced to the cortical penumbra of rats through the use of stereo-taxic techniques. Cerebral infarction size, cell apoptosis, and Bcl-2 protein expression were analyzed 24 hours after cerebral ischemia. 

MATERIALS AND METHODS

Design
Genetically engineered, randomized, controlled, animal study.
Time and setting
The experiment was performed at the Laboratory of Chongqing Medical University from May 2006 and January 2007.
Materials
Reagents and instruments are listed as follows:

A total of 54 healthy, male, adult Wistar rats, weighing 250–300 g, were provided by the Laboratory Animal Center of Chongqing Medical University (No. SCXK 2002 0001). All experimental procedures were in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Sci-ence and Technology of the People’s Republic of China[12]. The rats were randomly assigned to three groups (n = 18 for each group): normal saline, plasmid control, and recombinant Ngb (pCDNA3.1(+)/Ngb).
Methods
Plasmid extraction
Plasmids pCDNA3.1(+) and pCDNA3.1(+)/Ngb were extracted using a plasmid extraction kit and  identi-fied by BamHI and XhoI digestion[13-14]. Plasmid con-centration was determined by spectrophotometry[15], and the plasmids were stored in 1 μg/μL aliquots at –20 °C.
Stereotaxic transgene injection
Recombinant Ngb group: according to previously de-scribed methods[16], the rats were anesthetized by in-traperitoneal injection of 10% chloral hydrate    (0.35 mL/kg) and placed on the stereotaxic instrument. Ac-cording to The Rat Brain in Stereotaxic Coordinates[17], a medial incision, 3.0 cm posterior to the middle point of the ears, was made to expose the sagittal suture, ante-rior fontanel, and right cranium surface. A total of 1 μg/μL pCDNA3.1(+)/Ngb was drawn into a microsyringe (Eppendoff, Germany). Injection point 1 (3.8 mm right to sagittal suture and  1.7 mm anterior to the anterior fontanel) in the cranium was drilled with a dental drill, and the microsyringe needle was inserted 2.8 mm deep into injection point 1. pCDNA3.1(+)/Ngb (3.3 μL) was injected at 0.25 μL/min. The needle was withdrawn after 5 minutes, and the cranium hole was plugged with bone wax. A total of  3.3 μL pCDNA3.1(+)/Ngb (1 μg/μL) was injected into point 2 (3.8 mm right to the median line, 3.3 mm posterior to anterior fontanel, and 2.3 deep) using the above-described method.
Normal saline and plasmid control groups: A total of   3.3 μL normal saline and pCDNA3.1(+) (1 μg/μL) was injected into two animals using the above-described method.
Model of right middle cerebral arterial ischemia
At 24 hours following stereotaxic injection, the model of right middle cerebral arterial ischemia was established according to the modified thread method[18-19]. Briefly, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.35 mL/kg), and the right common carotid artery, as well as the right external carotid artery and its branches, were separated. The superior wall of the right common carotid artery was pierced with a 1-mL syringe needle, 1 cm to the distal common carotid artery, and a 0.25-mm nylon thread was inserted in the right internal carotid artery to a mean depth of (21.4 ±     1.2) cm.
A total of six animals from each group were selected at 24 hours following cerebral ischemia for TTC staining, six for immunofluorescence staining and in situ cell apop-tosis, and six for Western blot analysis.
TTC staining to determine cerebral infarction size
The rat brains were harvested and placed in 2% TTC solution at 37 °C in the dark for 30 minutes (infarction region remained white). The brain tissues were removed, coronally sectioned from anterior to posterior resulting in 2-mm thick sections, placed in 2% TTC solution at 37 °C in the dark for 20–30 minutes, and fixed in 4% parafor-maldehyde for 30 minutes. Each brain slice was imaged using a stereomicroscopy system. The brain slice and infarction areas were analyzed using a CMIAS medical imaging system according to the formula: V = (A1 + ? + An)t/2 (t = slice thickness, A = brain slice area or infarc-tion area)
Indirect immunofluorescence staining
Between injection points 1 and 2, brain tissues were prepared into paraffin sections. Some sections were selected, dewaxed, rinsed 3 times with 0.01 mol/L phosphate-buffered saline (PBS) for 10 minutes each, incubated in PBS containing 30 mg/L Triton X-100 at room temperature for 30 minutes, followed by goat se-rum for 12 hours at room temperature, rabbit anti- Bcl-2 polyclonal antibody (1: 1 000) for 24 hours at 4 °C, and FITC-labeled goat anti-rabbit IgG (1: 500) for 2 hours at room temperature in the dark. The sections were blocked with glycerine and PBS (1: 1), followed by  0.01 mol/L PBS (pH 7.4) rinsing after each step. The negative control was treated with PBS, rather than pri-mary antibody. Bcl-2 protein expression was observed in each section using laser confocal microscopy. The im-ages were digitally analyzed using imaging analysis software (LSM; Zeiss, Germany), and the Bcl-2-positive areas were determined at 24 hours following cerebral ischemia[5].
In situ cell apoptosis detection
The above-prepared paraffin sections were dewaxed, and cell apoptosis was detected using the TUNEL method[20-21]. Briefly, 6-μm thick sections were har-vested, attached to polylysine-treated coverslips, de-waxed, rinsed three times with 0.01 mol/L PBS for 10 minutes each, incubated with 50 μL pepsin K solution (10 μg/mL) at 37 °C for 10 minutes, and incubated with fresh methanol-prepared 0.03% H2O2 for 30 minutes to eliminate endogenous enzymes. The sections were rinsed three times with buffer A solution for 5 minutes each, placed in an humidity chamber with 50 μL TUNEL mixture on each section, and incubated at 37 °C for 30 minutes, followed by three wash steps with 0.01 mol/L PBS for 5 minutes each. The slides were immediately covered after adding   50 μL POD con-vert solution (Convert-POD) to each section and were incubated at  37 °C for 30 minutes, rinsed three times with PBS for 5 minutes each time, colored with DAB for 2 minutes, rinsed with tri-distilled water, lightly counterstained with hematoxylin, dehydrated, cleared, and mounted. The negative control was not incubated with TUNEL mixture or Converter-AP. High-power visual fields (× 200) with obvious apoptotic neurons in the ischemic penumbra were selected, and the mean number of apoptotic cells was quantified.
Western blot analysis
Brain tissues between injection points 1 and 2 were harvested, and protein was extracted[22] and stored at –80 °C. The protein was quantified following Coomassie brilliant blue G-250 staining, which was mixed with a four-fold volume of sample buffer solution and denatured at 95 °C for 5 minutes. Following 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, the mixture was electrotransformed to nitrocellulose filter. The bands were cut out according to molecular weight, incubated with rabbit anti-Bcl-2 polyclonal antibody (1: 300) and rabbit anti-rabbit β-actin monoclonal antibody (1: 400) at 4 °C overnight, rinsed, incubated with biotin-labeled goat anti-rabbit IgG serum (1: 100) at room temperature for 1.5 hours, and colored. With β-actin as an internal reference, the gray value of bands was determined using a CMIAS medical imaging system, and results were represented by the ratio of gray value to β-actin.
Main outcome measures
Cerebral infarction size, Bcl-2 expression, and apoptosis in the ischemic penumbra.
Statistical analysis
Measurement data from each group were expressed as Mean ± SD using SPSS 10.0 software package (SPSS, Chicago, IL, USA). Completely randomized, one-way analysis of variance was used for intergroup compari-sons, and the q-test for further pair comparisons. P < 0.05 was considered statistically significant.

RESULTS

Quantitative analysis of experimental animals
A total of 54 rats were included in the final analysis.
Plasmid identification
Ngb gene of Wistar rats was approximately 500 bp. The recombinant plasmid was digested by BamHI and XhoI (Figure 1).

Identification results showed that I and II were correct clones, and clone I was selected for recombinant plasmid extraction.
Cerebral infarction size
Compared with the normal saline and plasmid control groups, infarction size in the ischemic penumbra was significantly diminished following pCDNA3.1(+)/Ngb in-jection (P < 0.01), while no differences were determined between the normal saline and plasmid control groups (P > 0.05; Table 1, Figure 2).

Bcl-2 positive product area
Compared with the plasmid control group, the percentage of Bcl-2-positive cells in the ischemic penumbra was sig-nificantly increased following injection of recombinant Ngb (P < 0.01; Table 1, Figure 3).
Apoptosis detection
Compared with the plasmid control group, the number of apoptotic cells in the ischemic penumbra was significantly reduced following injection of recombinant Ngb (P < 0.01; Table 1, Figure 4).
Western blot analysis of Bcl-2 expression
Bcl-2 specific protein bands were observed with the pre-stained protein standards, revealing a molecular weight of 26 kD. The gray value comparison showed that Bcl-2 protein expression was upregulated by 40%–50% following injection of recombinant Ngb (P < 0.01, Table 1, Figure 5).

DISCUSSION

An understanding of the progression of cerebral ische-mia is essential for the treatment of ischemic brain dis-eases, and the establishment of an animal model of cerebral ischemia is a requirement for studies of ischemic brain diseases. Approximately 98% of rat genes are homologous to human genes, and rat cerebral vessels are structurally similar to humans. Moreover, a consistent ischemic range and reproducibility allows for the establishment of a rat model in cerebral ischemia studies[23]. Studies show varying results from rats of dif-ferent germ lines using the thread approach[24-25]. Therefore, in the present study, the thread was molded into an arc shape, allowing for consistent direction to-wards the internal carotid artery. A model of focal cere-bral ischemia was established, which was stable, reliable, reproducible, and exhibited a high success rate.
Ngb protein and its mRNA expression have been shown to increase after cerebral ischemia/hypoxia and play an important role in neuroprotection. Ngb mRNA increases 2–3-fold in cultured HN33 cells culture at 0.3% O2 for 24 hours[26]. Intracerebroventricular administration of Ngb antisense oligodeoxynucleotide in rats increases infarct volume and worsens functional neurological outcome, whereas intracerebral administration of a Ngb-expressing adeno-associated virus vector reduces infarct size and improves functional outcome following focal cerebral ischemia induced by occlusion of the mid-dle cerebral artery[8]. The present study directly injected pCDNA3.1(+)/Ngb into the cerebral ischemic penumbra, and infarct volume was reduced by 50% compared with the normal saline and plasmid control groups. This also indicated the neuroprotective effect of Ngb in focal cere-bral ischemia-induced nerve injury.
In addition, the effect of Ngb on cell apoptosis and Bcl-2 protein expression following focal cerebral ischemia identified possible anti-apoptotic mechanisms of Ngb. TUNEL staining selectively marks end products of apoptosis, i.e., double-strand DNA fragments, and has been used to determine whether Ngb inhibits neuronal apoptosis following cerebral ischemia[20]. Results from the present study showed that the number of apoptotic cells in the ischemic penumbra was significantly reduced at 24 hours following pCDNA3.1(+)/Ngb treatment, and Bcl-2 protein expression was remarkably increased, suggesting that Ngb inhibits apoptosis by upregulating Bcl-2 protein expression.
The Ngb gene vector, pCDNA3.1(+), avoids gene inser-tion mutation, carcinogenesis, or teratogenesis, which typically occurs when viruses serve as vectors[27]. Ngb decreases cellular apoptosis, which demonstrates that stereotaxically introduced recombinant Ngb into the brain inhibits cell apoptosis due to focal cerebral ischemia. Moreover, detection indexes of recombinant Ngb in the present study were similar to those of the normal saline and plasmid control groups, indicating negligible toxicity or side effects due to pCDNA3.1(+).
In conclusion, pCDNA3.1(+)-mediated Ngb expression increases Bcl-2 protein expression and inhibits cell apoptosis in the ischemic penumbra in a model of focal cerebral ischemia.

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 (Edited by Zhang XD, Zhou LX/Su LL/Song LP)