Osthole confers neuroprotection against cortical stab wound injury and attenuates secondary brain injury
- Yang Xia†1,
- Liang Kong†2,
- Yingjia Yao2,
- Yanan Jiao2,
- Jie Song2,
- Zhenyu Tao2,
- Zhong You1 and
- Jingxian Yang2Email author
© Xia et al. 2015
Received: 5 May 2015
Accepted: 16 August 2015
Published: 4 September 2015
Neuroendoscopy is an innovative technique for neurosurgery that can nonetheless result in traumatic brain injury. The accompanying neuroinflammation may lead to secondary tissue damage, which is the major cause of delayed neuronal death after surgery. The present study investigated the capacity of osthole to prevent secondary brain injury and the underlying mechanism of action in a mouse model of stab wound injury.
A mouse model of cortical stab wound injury was established by inserting a needle into the cerebral cortex for 20 min to mimic neuroendoscopy. Mice received an intraperitoneal injection of osthole 30 min after surgery and continued for 14 days. Neurological severity was evaluated 12 h and up to 21 days after the trauma. Brains were collected 3–21 days post-injury for histological analysis, immunocytochemistry, quantitative real-time PCR, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and enzyme-linked immunosorbent assays.
Neurological function improved in mice treated with osthole and was accompanied by reduced brain water content and accelerated wound closure relative to untreated mice. Osthole treatment reduced the number of macrophages/microglia and peripheral infiltrating of neutrophils and lowered the level of the proinflammatory cytokines interleukin-6 and tumor necrosis factor α in the lesioned cortex. Osthole-treated mice had fewer TUNEL+ apoptotic neurons surrounding the lesion than controls, indicating increased neuronal survival.
Osthole reduced secondary brain damage by suppressing inflammation and apoptosis in a mouse model of stab wound injury. These results suggest a new strategy for promoting neuronal survival and function after neurosurgery to improve long-term patient outcome.
Neuroendoscopy is a widely used neurosurgical option that employs a cylindrical retractor to access deep intracranial lesions . Both retractors and surgical procedures have been improved to facilitate and reduce the invasiveness of the procedure [2–4]; in recent years, commercial products such as BrainPath (NICO Corporation, 2014)  have been adopted to treat hard-to-reach areas. Nonetheless, while it is superior to traditional methods such as craniotomy, neuroendoscopy can nonetheless inflict damage to brain tissue.
During a typical neuroendoscopy procedure, mechanical trauma is caused by retractor insertion and includes the severance of capillaries, extracellular matrix, and glial and neuronal processes, leading to increased pressure region surrounding the retractor . Moreover, given that the retractor rests inside the brain for the duration of the surgery, brain pulsation can cause healthy tissue to beat against the rigid surface for up to several hours, inducing further injury . Finally, secondary brain injury may develop hours or even days later and is the main cause of delayed neuronal death after surgery [8, 9] and includes brain edema, reduction of regional blood flow, inflammation, apoptotic cell death, oxidative stress, and gliosis . Since primary brain injury caused by retractor insertion is irreversible, the main objective of medical treatment concerns the prevention and treatment of secondary brain injury .
The inflammatory response is a key factor in secondary injury following brain trauma and is induced by the release of proinflammatory cytokines [12, 13] that lead to the recruitment of peripheral leukocytes to the cerebral parenchyma and activation of resident immune cells [14, 15]. Neutrophils, monocytes, and lymphocytes modulate neuronal survival and death at the site of injury [14–17], while activated microglia release cytokines, reactive oxygen species, and other cytotoxic factors, further inducing neuronal death [16–18]. This response may be mitigated by suppressing inflammation with anti-inflammatory drugs. For example, the non-steroidal anti-inflammatory drug ibuprofen inhibits inflammation in mice following brain trauma by suppressing prostaglandin synthesis via cyclooxygenase 2 . Some herbs used in traditional medicine exert similar effects. Triptolide, an active ingredient of Tripterygium wilfordii Hook F, promotes the repair of injured spinal cord by inhibiting astrogliosis and inflammation , whereas the root of Panax ginseng C.A. Meyer (Araliaceae), also known as ginseng, inhibits interleukin (IL)-1β and IL-6, tumor necrosis factor (TNF)-α, and caspase-3 and B cell lymphoma (Bcl)-2-associated X protein (Bax) expression and stimulates IL-10, thereby suppressing inflammation and apoptotic cell death after traumatic brain injury .
Based on the above findings, we hypothesized that osthole can confer neuroprotection and attenuate the inflammatory response and reduce secondary damage in a mouse model of neuroendoscopy-induced brain injury. We investigated neurological function and inflammation following a 2-week treatment with osthole. The results suggest a new strategy for restoring neuronal function and improving long-term patient outcome after neuroendoscopy.
Materials and methods
Preparation of osthole
Osthole (catalog no. 110822-200305, purity >98 % (Fig. 1a) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) and dissolved in dimethyl sulfoxide (DMSO; <0.1 %), and stored at 4 °C [20, 21].
Animals, surgical procedures, and osthole administration
Adult C57BL/6J mice aged 3–4 months were housed under a 12:12-h light/dark cycle, with free access to food and water. Animal procedures conformed to guidelines set by Liaoning University of Traditional Chinese Medicine Institutional Animal Care and Use Committee, which are in accordance with those set by the National Institutes of Health (Bethesda, MD, USA). A mouse model of stab wound injury, as previously described in [29–31] with slight modifications, is created to mimic the neuroendoscopy procedure. Briefly, mice were anesthetized with ketamine/xylazine solution (50 mg/kg ketamine and 7.5 mg/kg xylazine in 0.9 % NaCl solution) and placed in a stereotaxic frame (ST-5ND-B, Chengdu, China). The head was shaved and the skin was disinfected with iodine/alcohol; body temperature was maintained at 37 °C throughout the surgical procedure using a heating pad . A midline incision was made through the scalp and the skin was retracted. A hole was made over the left cerebral hemisphere using a dental drill until the dura was exposed. A 20-gauge, 1.1-mm-diameter needle with a rigid core (BD Nexiva Closed IV Catheter System; BD Biosciences, Franklin Lakes, NJ, USA) was inserted at 2.5 mm lateral to the midline, 2.5 mm posterior to the lambdoidal suture, and at a depth of 2.5 mm from the surface of the brain (Fig. 1b). A blunt catheter needle with a sharp core was inserted into the mouse brain. Upon reaching the target area, the needle was left in place for 20 min while the core was removed immediately. This procedure was designed to mimic neuroendoscopy. The injury site was then covered with sterile bone wax, the skin incision was closed with sutures, and the mouse was allowed to recover in its cage .
Mice were randomly divided into five groups: groups 1–3 (SWI+Ost) were administered osthole by i.p. injection 30 min after surgery at 10, 20, and 30 mg/kg, respectively, dissolved in 0.1 % DMSO and phosphate buffered saline (PBS) [11, 27] followed by once daily injections for the next 14 days (n = 30, 30, and 54 for groups 1, 2, and 3, respectively); mice in the SWI control group were given 0.1 % DMSO in PBS by i.p. injection (200 μl) each day for 14 days (n = 54), and naive C57BL/6J mice were used as controls (n = 54). Mice were sacrificed between 3 and 21 days post-injury (dpi) for analyses.
Assessment of neurological function
Neurological function was assessed with a modified neurological severity score (NSS) at 12 h and 3, 7, 14, and 21 dpi, as previously described (n = 6 per group for each time point) [29, 34, 35]. The evaluation consisted of motor (muscle status and abnormal movement), sensory (visual, tactile, and proprioceptive), reflex, and balance tests, with results measured on a scale of 0–18 (0 = normal, 1–6 = mild injury, 7–12 = mean-moderate injury; 13–18 = severe injury, and 18 = maximal deficit) and the total score representing the sum of all test scores. One point was awarded for the inability to perform a test or lack of a tested reflex; therefore, a higher score indicated a greater degree of injury. The test was administered by blinded, trained investigators, and mice were familiarized with the testing environment before being subjected to brain injury.
Measurement of brain water content
Brain water content was measured 72 h after SWI (n = 6 per group). Following anesthesia and decapitation, brains were dissected, separated along the midline, and the cerebellum was removed. Ipsilateral hemisphere wet weight was obtained on a pre-weighed piece of aluminum foil; after drying in an electric oven at 100 °C for 24 h, the percent water was calculated as (wet weight − dry weight)/(wet weight) [11, 36, 37].
Hematoxylin and eosin staining and measurement of lesion size
At 3, 7, 14, and 21 dpi, mice were anesthetized and transcardially perfused with 4 % paraformaldehyde in cold phosphate buffer (n = 6 per group for each time point). Brains were immediately dissected and fixed in 10 % buffered formalin and embedded in paraffin. Serial coronal section 10 μm thick were cut in a direction parallel to the needle penetration line at 100-μm intervals so as to cover the entire lesion site. Sections were mounted on glass slides for hematoxylin and eosin (H & E) staining [37, 38] and visualized on a Nikon Eclipse E800 microscope (Tokyo, Japan) with a digital camera. Changes in cytoarchitecture were analyzed, and the size of the wound cavity was measured in each section by tracing a line across the top and along the edge of the tissue lining the lesion with ImageJ software (National Institutes of Health) [13, 18]. Measurements were taken from six sections per mouse from six mice at each time point [30, 39, 40].
Brains were flash-frozen in cold isopentane on dry ice immediately following perfusion and stored at −80 °C. Serial frozen sections were cut at a thickness of 8 μm on a cryostat microtome (Leica, Nussloch, Germany) and fixed with 4 % paraformaldehyde in PBS for 30 min. After washing twice in PBS, endogenous peroxidase activity was quenched by incubation in 3 % hydrogen peroxide/0.1 % Triton X-100 for 15 min at room temperature, followed by two washes in PBS and blocking with 10 % goat serum for 30 min. Sections were incubated at 4 °C overnight with antibodies against the following proteins: neurofilament (NF)-M, neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP) (all at 1:150, from StemCell Technologies, Vancouver, Canada), ionized calcium-binding adaptor molecule (Iba)-1 (1:100), caspase-3 (1:150), and myeloperoxidase (MPO) (1:150) (all from Abcam, Cambridge, MA, USA). After three PBS washes, sections were incubated with appropriate fluorescein isothiocyanate- or Cy3-conjugated secondary antibodies (1:200; Jackson ImmunoResearch Lab, West Grove, PA, USA) for 60 min at room temperature and counterstained with 4′6-diamidino-2-phenylindole (DAPI), followed by three PBS washes. Sections were mounted with mounting medium (Vector Laboratories, Burlingame, CA, USA) and visualized using the Nikon Eclipse E800 microscope.
Cells expressing specific antigen were counted using ImageJ software [20, 31, 41–45] in six non-adjacent brain sections from six mice per group in five digital images of each section acquired with the same exposure parameters. The expression level was measured by pixel intensity as previously reported [44, 45]. Pixel intensity was measured from areas immediately surrounding the lesion (Fig. 1b). The total number of DAPI+ nuclei was similar between measured areas (n = 6 for each test).
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
Brain sections from mice at 7 dpi were analyzed for apoptotic cells using the Fluorescence In Situ Cell Death Detection kit (Roche, Chicago, IL, USA) according to the manufacturer’s instructions (n = 6 per group). The number of TUNEL-positive cells in each section in areas surrounding the lesion was counted in six sections per mouse and six mice per group using ImageJ software [9, 29, 39].
Quantitative real-time (RT-) PCR
Brains were collected at 3 and 7 dpi (n = 6 per group for each time point), and punch biopsies (diameter: 5 mm) of the injured cortex were obtained. Tissue samples were rinsed with diethylpyrocarbonate-treated water, placed in 2-ml Eppendorf tubes, and stored at −80 °C for RT-PCR. Total RNA was extracted from the tissue with TRIzol reagent and reverse transcribed to cDNA using a RevertAid First Strand cDNA Synthesis kit (Thermo Scientific, Vilnius, Lithuania) . The PCR reaction (35 cycles) was carried out using a DreamTaq Green PCR Master Mix Kit (Thermo Scientific). Quantitative RT-PCR was performed using the following forward and reverse primer sets designed using Premier Biosoft 5 (Palo Alto, CA, USA): Bax, 5′-CTG ACA TGT TTT CTG ACG GC-3′ and 5′-TCA GCC CAT CTT CTT CCA GA-3′; Bcl-2, 5′-CGC TGG GAG AAC AGG GTA-3′ and 5′-GGG CTG GGA GGA GAA GAT-3′; caspase-3, 5′-AGA TAC CGG TGG AGG CTG ACT-3′ and 5′-TCT TTC GTG AGC ATG GAC ACA-3′; IL-6, 5′-AGC CAG AGT CCT TCA GAG AG-3′ and 5′-TCC TTA GCC ACT CCT TCT GT-3′; and β-actin (control), 5′-GGG AAA TCG TGC GTG ACA T-3′ and 5′-TCA GGA GGA GCA ATG ATC TTG-3′. Products were resolved by 1.5 % agarose gel electrophoresis with ethidium bromide staining. The mRNA level of IL-6 was detected at 3 dpi, and those of Bax, Bcl-2, and caspase-3 were detected at 7 dpi. Quantitative analysis was performed using a Tanon 4100 Gel Imaging System (Tanon Science & Technology Co., Shanghai, China).
Analysis of cytokine levels by enzyme-linked immunosorbent assay (ELISA)
Brains were collected at 3 dpi, and punch biopsies (diameter: 5 mm) of the injured cortex were obtained and stored at −80 °C until use (n = 6). Brain homogenates were centrifuged at 4 °C and 15,000 rpm for 20 min, and the supernatant was transferred to an Eppendorf tube. IL-6 and TNF-α levels in each sample were measured using ELISA kits (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions on a fluorescent plate reader at 450 nm .
Data were analyzed using SPSS version 13.0 (SPSS, Chicago, IL, USA) and are presented as mean ± standard deviation. Differences between groups were assessed by one-way analysis of variance, and post-hoc multiple comparisons were carried out with the Student-Newman-Keuls test. P < 0.05 was considered statistically significant.
In this study, we used naive (uninjured) mice as controls rather than those subjected to craniotomy. It was previously shown that craniotomy is equivalent to a minor injury in terms of the acute inflammatory response that is induced [9, 47]; mice that underwent craniotomy had similar numbers of GFAP-positive astrocytes to those that experienced moderate cortical impact injury .
Osthole treatment improves neurological function after SWI
Increase in brain water content caused by injury is reversed by osthole treatment
Cerebral edema is caused by damage to the blood-brain barrier (BBB) after traumatic injury, and the resultant increase in brain water content is a marker of brain damage . Brain edema worsened over time, peaking at 72 h before declining thereafter . At 3 dpi, brain water content was higher in the SWI than in the naïve control group (85.27 % ± 4.32 % vs. 57.56 % ± 6.45 %) (Fig. 2b); this was reduced in a dose-dependent manner by treatment with osthole (69.45 ± 9.26; P < 0.01 vs. SWI control).
Osthole accelerates the closure of the brain cavity resulting from injury
Osthole attenuates the inflammatory response in the injured brain
Osthole promotes neuronal survival and reduces apoptosis in the injured brain
In this study, we mimicked the procedure of neuroendoscopy by using a needle to create a stab wound in the mouse brain and investigated whether osthole treatment can protect against secondary brain damage. Osthole treatment (30 mg/kg i.p. once daily for 14 days) reduced the number of microglia/macrophages in the brain parenchyma, decreased the number of peripheral infiltrating leukocytes at the lesion, reduced proinflammatory cytokine level, and inhibited apoptosis. These effects were exerted via downregulation of IL-6, TNF-α, and caspase-3 expression and a reduction in the ratio of Bax to Bcl-2. Thus, osthole can prevent secondary brain damage through anti-inflammatory as well as anti-apoptotic mechanisms.
Traumatic injury to the brain triggers an inflammatory response characterized by activation of microglia and invasion of peripheral macrophages, resulting in secondary tissue damage at the injury site [19, 52]. The lack of spontaneous tissue repair is due not only to the inability of neurons to proliferate but also to the lesion microenvironment, which contains many toxic factors [53, 54]. Accordingly, the goal of treatment is to suppress inflammation during the acute phase so as to improve the environment for and prevent secondary damage to surviving neurons.
Osthole has anti-inflammatory, anti-immunomodulatory, anti-apoptotic, and anti-oxidative stress as well as neurotrophic effects [20–24]. It also protects against cerebral ischemic injury by reducing oxidative stress injury and preserving the integrity of the BBB . Our previous study showed that osthole treatment reduced clinical severity in EAE mice by suppressing the autoimmune response, decreasing IL-17 and IFN-γ production, and increasing neurotrophic support, thereby creating an environment favorable to neuronal survival, protecting the myelin sheath from demyelination, and promoting the repair of myelin/axons , as reported by others . In the present study, mice treated with osthole exhibited a low NSS, which was accompanied by a significant reduction in brain water content and accelerated closure of the wound cavity as compared to untreated control animals. Moreover, osthole treatment following injury was also associated with a lower number of microglia/macrophages and neutrophils at the injury site and reduced levels of the proinflammatory cytokines IL-6 and TNF-α. The effects of osthole also included inhibition of apoptosis, which is mediated in part by caspase-3 activation [56–58]. Thus, our results suggest that osthole can prevent post-injury inflammation and apoptosis to reduce secondary injury to brain tissue, creating an environment conducive to functional recovery.
A previous study has shown that initial needle penetration results in an immediate loss of tissue and the formation of a cavity that gradually increases in size, reaching a maximum size 3 days after injury, at which time peak rates of apoptosis around the lesioned area and robust astroglial reactivity are also observed . The extent of brain tissue damage was dependent on the mechanics of needle insertion; for example, high insertion force and speed produced greater tissue damage . As expected, the cavity volume was larger than the diameter of needle that was used. The cavity shrank over time as the tissue healed, which was consistent with previous reports [59, 60].
NFs are the most abundant cytoskeletal protein in large myelinated axons [40, 61]; specifically, NF-M is important for the stabilization of mature axons . We observed a decrease in NF-M expression in injured animals, indicating axonal loss. In contrast, mice treated with osthole showed increased NF-M expression, providing structural evidence for the effects of osthole in promoting neuronal restoration in the injured brain.
IL-6 is a major inducer of immune and inflammatory responses under conditions of injury  and enhances glutamate-mediated excitotoxicity in cerebellar granule cells in vitro and causes damage to the BBB in vivo . Transgenic mice overexpressing IL-6 display gliosis, neuronal loss, and learning disabilities with prominent neurodegeneration . Hence, excessive IL-6-mediated inflammation is likely involved in the unfavorable outcomes associated with SWI. Osthole treatment partly blocked the increase in IL-6 gene expression resulting from injury, providing additional evidence that osthole suppresses neuroinflammation via downregulation of IL-6.
The Bcl-2 family includes genes encoding the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2 [66, 67]. Bcl-2 overexpression inhibits neuronal apoptosis and stimulates the recovery of neurological function [68, 69], while overexpressing Bax induces apoptosis . Bax upregulation and Bcl-2 downregulation increases the Bax to Bcl-2 ratio; this may be directly associated with cytochrome c release  and increased expression of caspase-3, which induces apoptosis . In the present study, osthole treatment reduced the Bax to Bcl-2 ratio and caspase-3 level that were elevated by SWI, providing insight into the mechanism underlying the anti-apoptotic effects of osthole.
Osthole treatment conferred neuroprotection and reduced tissue damage in an experimental cortical SWI model, reducing secondary brain damage via anti-inflammatory and -apoptotic mechanisms. These findings demonstrate that osthole has therapeutic potential for reducing injury-induced neuroinflammation to improve long-term patient outcome after neuroendoscopic surgery.
This work was supported by the National Natural Science Foundation of China (grant No. 81173580).
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