Improvement in functional recovery with administration of Cerebrolysin after experimental closed head injury

Laboratory investigation

Restricted access

Object

Cerebrolysin is a unique peptide preparation that mimics the action of neurotrophic factors. This study was designed to investigate the effects of acute treatment of experimental closed head injury (CHI) in rats with Cerebrolysin on neurological function.

Methods

Adult male Wistar rats (n = 60) were subjected to impact acceleration–induced CHI. Closed head injured rats received intraperitoneal injection of saline (n = 30) or Cerebrolysin (2.5 ml/kg, n = 30) starting 1 hour postinjury and administered once daily until they were killed (2 or 14 days after CHI). To evaluate functional outcome, the modified neurological severity score (mNSS), foot fault, adhesive removal, and Morris water maze (MWM) tests were performed. Animals were killed on Day 14 (n = 20) after injury, and their brains were removed and processed for measurement of neuronal cells, axonal damage, apoptosis, and neuroblasts. The remaining rats (n = 40) were killed 2 days postinjury to evaluate cerebral microvascular patency by fluorescein isothiocyanate (FITC)–dextran perfusion (n = 16) and to measure the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase–9 (MMP-9) by using real-time reverse transcriptase-polymerase chain reaction (RT-PCR, n = 8) and by immunohistochemical analysis (n = 16).

Results

At 14 days post-CHI, the Cerebrolysin treatment group exhibited significant improvements in functional outcomes (the adhesive removal, mNSS, foot-fault, and MWM tests), and significantly more neurons and neuroblasts were present in the dentate gyrus (DG) (p < 0.05) compared with the saline-treated group (p < 0.05). At 2 days post-CHI, the Cerebrolysin group exhibited a significantly higher percentage of phosphorylated neurofilament H (pNF-H)–positive staining area in the striatum (p < 0.05), a significant increase in the percentage of FITC-dextran perfused vessels in the brain cortex (p < 0.05), a significant increase in the number of VEGF-positive cells (p < 0.05), and a significant reduction in the MMP-9 staining area (p < 0.05) compared with the saline-treated group. There was no significant difference in mRNA levels of MMP-9 and VEGF in the hippocampus and cortex 48 hours postinjury between Cerebrolysin- and saline-treated rats that sustained CHI.

Conclusions

Acute Cerebrolysin treatment improves functional recovery in rats after CHI. Cerebrolysin is neuroprotective for CHI (increased neurons in the dentate gyrus and the CA3 regions of the hippocampus and increased neuroblasts in the dentate gyrus) and may preserve axonal integrity in the striatum (significantly increased percentage of pNF-H–positive tissue in the striatum). Reduction of MMP-9 and elevation of VEGF likely contribute to enhancement of vascular patency and integrity as well as neuronal survival induced by Cerebrolysin. These promising results suggest that Cerebrolysin may be a useful treatment in improving the recovery of patients with CHI.

Abbreviations used in this paper:Ang1 = angiopoietin 1; BBB = blood-brain barrier; BDNF = brain-derived neurotrophic factor; CHI = closed head injury; CTNF = ciliary neurotrophic factor; DG = dentate gyrus; FITC = fluorescein isothiocyanate; GDNF = glial cell–derived neurotrophic factor; IGF = insulin-like growth factor; miRNA = microRNA; MMP-9 = matrix metalloproteinase–9; mNSS = modified Neurological Severity Score; MWM = Morris water maze; pNF-H = phosphorylated neurofilament H; RT-PCR = reverse transcriptase-polymerase chain reaction; SGZ = subgranular zone; TBI = traumatic brain injury; TUNEL = terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling; VEGF = vascular endothelial growth factor.

Article Information

Address correspondence to: Ye Xiong, M.D., Ph.D., Department of Neurosurgery, Henry Ford Health System, E&R Building, Room #3096, 2799 West Grand Boulevard, Detroit, Michigan 48202. email: yxiong1@hfhs.org.

Please include this information when citing this paper: published online April 12, 2013; DOI: 10.3171/2013.3.JNS122061.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Representative coronal brain sections with NeuN staining showing fields selected for quantitative measurement of cells and structure. Left: Section encompassing the striatum. Right: Section encompassing the hippocampus. Bar = 2 mm.

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    Cerebrolysin effect on functional outcome. A: Cerebrolysin treatment significantly reduces adhesive removal time from Days 7 to 14 after CHI compared with saline treatment. B: Cerebrolysin treatment reduces the frequency of foot faults from Days 1 to 14 after CHI compared with the saline treatment. C: Cerebrolysin significantly lowers the mNSS scores from Days 7 to 14 compared with the saline group. D: Cerebrolysin treatment significantly improves spatial learning performance compared with the saline group from Days 11 to 14 after CHI. E: Cerebrolysin treatment significantly reduces time to reach the hidden platform in the MWM compared with the saline group from Days 11 to 14 after CHI. F: There is no significant difference in swim speed between the groups, indicating that motor deficits did not contribute to cognitive deficits. There were 10 rats/group. D = day; Pre = preinjury. Data represent the mean ± SD. *p < 0.05 vs saline.

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    Cerebrolysin effect on neuronal loss after CHI. Left: NeuN staining. Cerebrolysin significantly reduces neuronal cell loss in the DG and CA3 region examined at 14 days compared with the saline-treated group. Right: Bar graph showing the number of neuronal cells. There were 10 rats/group. CT = cortex; ST = striatum; TH = thalamus. Bar = 50 μm. *p < 0.05 vs saline. Data represent the mean ± SD.

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    Cerebrolysin effect on axonal damage in the striatum 14 days after CHI. Left: Phosphorylated neurofilament H staining. Compared with the saline-treated group, Cerebrolysin treatment significantly increases pNF-H-positive area in the striatum. Right: Bar graph showing the percentage of the pNF-H-positive area. There were 10 rats/group. CC = corpus callosum. Bar = 50 μm. *p < 0.05 vs saline. Data represent the mean ± SD.

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    Cerebrolysin effect on apoptosis. TUNEL staining was performed to detect apoptotic cells 14 days after CHI. There is no significant difference in the number of apoptotic cells detected between the saline-treated and Cerebrolysin-treated groups. There were 10 rats/group. Bar = 50 μm. Data represent the mean ± SD.

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    Cerebrolysin effect on neuroblasts in the DG 14 days after CHI. Upper: DCX staining was used to detect neuroblasts in the DG. Compared with the saline-treated group, Cerebrolysin treatment significantly increases the DCX-positive neuroblasts in the DG. Lower: Bar graph showing the number of DCX-positive cells. There were 10 rats/group. Bar = 50 μm. Data represent the mean ± SD.

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    Cerebrolysin (Cereb) effect on the patency of cerebral microvessels. FITC-dextran was administered intravenously to rats 2 days after CHI. The cortex tissue from saline-treated CHI rats is darkened because of low blood perfusion, that is, low fluorescence (A). Compared with the saline-treated group, Cerebrolysin treatment significantly increases the FITC-dextran–perfused area in the cortex (B). Bar graph showing the percentage of FITC-dextran-perfused area (C). There were 8 rats/group. Hippo = hippocampus. Bar = 50 μm. Data represent the mean ± SD.

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    Cerebrolysin effect on MMP-9 expression after CHI. Left: MMP-9 may degrade the integrity of the BBB. Compared with the saline-treated group, Cerebrolysin treatment significantly decreases MMP-9 expression after CHI in many brain regions. Right: Bar graph showing the percentage of the MMP-9–positive area. There were 8 rats/group. Bar = 50 μm. Data represent the mean ± SD.

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    Cerebrolysin effect on VEGF expression after CHI. Left: To detect the effect of Cerebrolysin on promoting VEGF expression, VEGF staining was performed. Compared with the saline-treated group, Cerebrolysin treatment significantly increases VEGF-positive cells after CHI in many brain regions. Right: Bar graph showing the number of VEGF-positive cells. There were 8 rats/group. Bar = 50 μm. Data represent the mean ± SD.

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    Cerebrolysin effect on mRNA levels of Ang1, MMP-9, and VEGF after CHI. To examine the effect of Cerebrolysin on expression of genes that mediate microvascular integrity and neuronal damage, mRNA levels of Ang1, VEGF, and MMP-9 were measured by real-time RT-PCR analysis in the cortex and hippocampus. No significant difference in mRNA levels of these genes was detected between the saline-treated and Cerebrolysin-treated groups. There were 4 rats/group.

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