Randomized controlled trial of Cerebrolysin’s effects on long-term histological outcomes and functional recovery in rats with moderate closed head injury

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OBJECTIVE

The authors previously demonstrated that Cerebrolysin is effective for treatment of mild closed head injury (CHI) when administered 4 hours after injury. The aim of this study was to determine Cerebrolysin’s effects on functional and histological outcomes in rats subjected to moderate CHI.

METHODS

In this randomized, blinded, and vehicle-controlled preclinical trial, male adult Wistar rats subjected to moderate CHI received either Cerebrolysin treatment at a dose of 2.5 ml/kg (n = 13) or vehicle (saline, n = 13) intraperitoneally administered daily for 10 days, starting at 4 hours after injury. Animals were subjected to cognitive and sensorimotor functional tests at multiple time points, and they were killed 3 months after injury. The brains were processed for analyses of neuronal cell loss, amyloid precursor protein, axonal damage, and neurogenesis.

RESULTS

Compared with rats treated with vehicle (saline), rats treated with Cerebrolysin had significantly increased numbers of neuroblasts and newborn mature neurons in the dentate gyrus (DG) and attenuated amyloid precursor protein accumulation and axonal damage in various brain regions, as well as decreased neuronal loss in the DG and cornu ammonis 3 (CA3) region of the hippocampus (p < 0.05). Global testing using generalized estimating equations showed a significant beneficial effect of Cerebrolysin treatment on sensorimotor functional outcomes from 1 day to 3 months after injury compared to that of saline treatment (p < 0.05). Compared with vehicle-treated rats, Cerebrolysin-treated rats showed significantly and robustly improved long-term (up to 3 months) cognitive functional recovery, as measured by social interaction, Morris water maze, novel object recognition, and odor recognition tests. In the Cerebrolysin-treated rats there were significant correlations between multiple histological outcomes and functional recovery evident 3 months after moderate CHI, as indicated by Pearson partial correlation analyses.

CONCLUSIONS

The authors’ findings demonstrate that Cerebrolysin treatment significantly improves long-term functional and histological outcomes in rats with moderate CHI, with functional outcomes significantly correlated with histological indices of neuroplasticity and neuroprotection. These data indicate that Cerebrolysin may be useful for the treatment of moderate CHI.

ABBREVIATIONS AD = Alzheimer’s disease; APP = amyloid precursor protein; BrdU = bromodeoxyuridine; BSA = bovine serum albumin; CA = cornu ammonis; CC = corpus callosum; CHI = closed head injury; CT = cortex; DCX = doublecortin; DG = dentate gyrus; IP = intraperitoneal/intraperitoneally; mNSS = modified neurological severity score; MWM = Morris water maze; NeuN = neuronal nuclei; NOR = novel object recognition; PBS = phosphate-buffered saline; pNfH = phosphorylated neurofilament heavy subunit; TBI = traumatic brain injury.

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Article Information

Correspondence Ye Xiong: Henry Ford Health System, Detroit, MI. yxiong1@hfhs.org.

INCLUDE WHEN CITING Published online September 6, 2019; DOI: 10.3171/2019.6.JNS191027.

Disclosures This work was funded by EVER Neuro Pharma GmbH.

The authors disclose receipt of the following financial support for the research, authorship, and/or publication of this article. Dr. Chopp received travel support and honoraria from EVER Neuro Pharma GmbH for presenting data at scientific meetings. EVER Neuro Pharma GmbH also provided financial support to Dr. Chopp for travel to meetings supported by EVER Neuro Pharma GmbH. Dr. Mei Lu and Ms. Talan Zhang received some salary support provided by EVER Neuro Pharma GmbH for statistical analyses. Drs. Yanlu Zhang and Ye Xiong had a percentage of salary support provided from a nonrestricted research contract with EVER Neuro Pharma GmbH.

© AANS, except where prohibited by US copyright law.

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Figures

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    Effects of Cerebrolysin on sensorimotor functional outcomes measured by the mNSS (A), adhesive-removal (B), and footfault (C) tests and on spatial learning performance in the MWM test (D–F) 3 months after moderate CHI. A: Cerebrolysin treatment significantly decreased mNSS compared to vehicle treatment from day 7 to 3 months. B: Cerebrolysin treatment significantly reduced adhesive-removal times compared with vehicle treatment from day 7 to 3 months. C: Cerebrolysin treatment significantly reduced the frequency of footfaults compared with vehicle treatment from day 1 to 3 months. D: Moderate CHI did not affect the swim speed among all groups. E: Cerebrolysin treatment significantly increased the time spent in the correct quadrant compared with vehicle treatment on days 87, 88, 89, and 90. F: Cerebrolysin treatment significantly reduced the time to reach the hidden platform in the MWM compared to vehicle treatment on days 86, 87, 88, and 90. *p < 0.05 versus vehicle. Data are presented as mean ± SD; n = 10 in sham group, n = 13 in vehicle group, and n = 13 in treatment group. Cereb = Cerebrolysin; D = day; Rx = Cerebrolysin or vehicle treatment; 4h, daily = IP administration of Cerebrolysin or vehicle was performed in rats starting at 4 hours after TBI, and then daily for 10 days. Figure is available in color online only.

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    Effects of Cerebrolysin on social interaction in the 3-chamber test (A), NOR memory test (B), and odor-based novelty recognition memory test (C) 3 months after moderate CHI. A: Compared with the vehicle-treated rats, the Cerebrolysin-treated rats spent significantly more time exploring the novel rat than the familiar rats. B: Compared with the vehicle-treated rats, the Cerebrolysin-treated rats spent significantly more time exploring the novel object than the familiar object. C: Compared with the vehicle-treated rats, the Cerebrolysin-treated rats spent significantly more time exploring the novel-odor bead than the familiar bead. *p < 0.05 versus vehicle. Data are presented as mean ± SD; n = 10 rats in sham group, n = 13 in vehicle group, and n = 13 in treatment group. Figure is available in color online only.

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    Effects of Cerebrolysin on brain tau accumulation 3 months after moderate CHI. Compared with the vehicle-treated group (B, E, and H), the Cerebrolysin-treated group (C, F, and I) had significantly less tau accumulation in the CA3 and CT brain regions. Data on tau accumulation in each area are shown in the bar graph (J). Sham group (A, D, and G). Bar = 25 μm. *p < 0.05 versus vehicle. Data are presented as mean ± SD; n = 10 rats in sham group, n = 13 in vehicle group, and n = 13 in treatment group. Figure is available in color online only.

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    Effects of Cerebrolysin on the number of surviving cells 3 months after moderate CHI. Compared with the vehicle-treated group (B, E, and H), the Cerebrolysin-treated group (C, F, and I) had significantly more surviving neuron cells in the CA3 and DG regions. Sham group (A, D, and G). The data on the number of NeuN+ cells are shown in the bar graph (J). Bar = 50 μm. *p < 0.05 versus vehicle. Data are presented as mean ± SD; n = 10 rats in sham group, n = 13 in vehicle group, and n = 13 in treatment group. Figure is available in color online only.

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    The effects of Cerebrolysin on brain APP accumulation 3 months after moderate CHI. Compared with the vehicle-treated group (B, E, and H), the Cerebrolysin-treated group (C, F, and I) had significantly less APP accumulation in the CT, CA3, and DG regions. Sham group (A, D, and G). The data on the APP+ area are shown in the bar graph (J). Bar = 25 μm. *p < 0.05 versus vehicle. Data are presented as mean ± SD; n = 10 rats in sham group, n = 13 in vehicle group, and n = 13 in treatment group. Figure is available in color online only.

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    Effects of Cerebrolysin on pNfH+ areas 3 months after moderate CHI. Compared with the vehicle-treated group (B, E, and H), the Cerebrolysin-treated group (C, F, and I) had significantly increased pNfH+ areas in CT, CA3, and DG brain regions. Sham group (A, D, and G). The data on the pNfH+ area are shown in the bar graph (J). Bar = 25 μm. *p < 0.05 versus vehicle. Data are presented as mean ± SD; n = 10 rats in sham group, n = 13 in vehicle group, and n = 13 in treatment group. Figure is available in color online only.

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    Effects of Cerebrolysin on the number of DCX+ neuroblasts 3 months after moderate CHI. Compared with the vehicle-treated group (B), the Cerebrolysin-treated group (C) had significantly increased DCX+ neuroblasts in the DG. Sham group (A). The data on the number of DCX+ cells are shown in the bar graph (D). Bar = 25 μm. *p < 0.05 versus vehicle. Data are presented as mean ± SD; n = 10 rats in sham group, n = 13 in vehicle group, and n = 13 in treatment group. Figure is available in color online only.

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    Effects of Cerebrolysin on the number of BrdU/NeuN-positive newborn mature neurons in the DG 3 months after moderate CHI. Compared with the vehicle-treated group (B), the Cerebrolysin-treated group (C) had significantly increased newborn mature neurons identified with BrdU/NeuN+ cells in the DG (arrows). Sham group (A). The data on the number of BrdU/ NeuN+ cells are shown in the bar graph (D). Bar = 25 μm. *p < 0.05 versus vehicle. Data are presented as mean ± SD; n = 10 rats in sham group, n = 13 in vehicle group, and n = 13 in treatment group. Figure is available in color online only.

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