Neuroprotective effect of Da Chuanxiong Formula against cognitive and motor deficits in a rat controlled cortical impact model of traumatic brain injury

Abstract

Ethnopharmacological relevance

Da Chuanxiong Formula (DCXF) is one of the famous herb pairs that contains dried rhizomes of Ligusticum chuanxiong Hort. and Gastrodia elata Bl. in the mass ratio of 4:1. This classic representative traditional Chinese medicine has been widely used to treat brain diseases like headache and migraine caused by blood stasis and wind pathogen. However, the therapeutic effect of DCXF on traumatic brain injury (TBI) has not been reported yet.

Aim of study

The present study was performed to investigate the neuroprotective effects of DCXF and its underlying mechanisms in the controlled cortical impact (CCI)-induced TBI rat model.

Materials and methods

Male Sprague-Dawley rats were divided into four groups: Sham, TBI control, 1X DCXF (520.6 mg/kg) and 5X DCXF (2603.0 mg/kg). Two treatment groups (1X and 5X DCXF) were intragastrically administered daily for 7 days before CCI-induced TBI and then DCXF treatments were continued post-TBI until the animal behavioral tests, including Morris water maze test, acceleration rotarod motor test and CatWalk quantitative gait analysis test, were done. The brain water content and blood brain barrier (BBB) integrity were measured by wet-dry weight method and Evans blue method, respectively. The number of neuron cells, neural stem cells (NSCs), GFAP positive cells (astrocyte) as well as Iba-1 positive cells (microglia) were determined by histology and immunohistochemistry.

Results

Treatment with DCXF significantly improved the learning ability and memory retention in Morris water maze test, and remarkably enhanced motor performances in acceleration rotarod motor test and catwalk quantitative gait analysis test after TBI. Moreover, DCXF treatment was able to reduce BBB permeability, brain edema, microglia and astrocyte activation, improve the proliferation of NSCs and decrease neurons loss in the brain with TBI.

Conclusions

The present study demonstrated that DCXF treatment could decrease BBB leakage and brain edema, reduce neuron loss, microglia and astrocyte activation, and increase NSCs proliferation, which may contribute to the cognitive and motor protection of DCXF in the TBI rats. It is the first time to provide potentially underlying mechanisms of the neuroprotective effect of DCXF on TBI-induced brain damage and functional outcomes.

Introduction

Traumatic brain injury (TBI) is an injury caused by a bump, blow, jolt or penetrating wound to the head, which disrupts the normal function of the brain included cognitive, behavioral and psychosocial deficits. Road traffic accidents, falls, violence, work and sports related injuries are the major etiologies of traumatic brain injury. According to the World Health Organization, the global incidence rate of TBI is likely 600 per 100,000 annually. TBI is predicted to become one of the major causes of death and disability by the year 2020 (Hyder et al., 2007). In China, the average annual number of TBI cases is 3–4 million (Liu, 2015). The highest incidence of TBI are found in young people at age 19 or younger and older adults over 75 years, predominantly males (Leo and McCrea, 2016). The annual economic cost included lifetime medical costs and productivity losses of TBI in the United States is estimated to be more than $60 billion (Brown et al., 2008). In addition, patients with TBI increased the risk to develop neurodegenerative disorders such as Alzheimer disease, chronic traumatic encephalopathy, amyotrophic lateral sclerosis and Parkinson disease due to its multiple and multidirectional biochemical events (Gardner et al., 2015, Gupta and Sen, 2016). Overall, TBI is a critical public health problem worldwide, which severely affects the quality of the affected person's daily life and creates a financial burden for families and societies (Faul and Coronado, 2015).

TBI is a highly complex process and manifested in functional deficits due to both primary and secondary mechanisms. Primary injury refers to the immediate mechanical damage at the time of injury, resulting in necrotic cell death, shearing and tearing of blood vessels, neurons, glia and axons, and initiation of secondary injury. Secondary injury evolves over hours to days, even months, following the primary injury. It is the consequence of ongoing metabolic, cellular and molecular changes which cause further damage. Both primary craniocerebral injury and secondary lesions can induce oxidative stress, inflammation, brain edema, blood-brain barrier (BBB) damage and eventually result in neuronal cell death and functional deficits (Xiong et al., 2013). Among of these mechanisms, neuroinflammation is widely consider as a vital factor which contributes to the initiation and progression of the secondary phase of TBI, leading to the widespread cell death, suppression of NSCs proliferation and neurogenesis, and cortical as well as hippocampal neurodegeneration. The inflammatory responses are regulated by activated astrocyte and microglia, along with infiltrating macrophage, which are able to upregulate and secrete many inflammatory mediators such as cytokines, nitric oxide (NO) and chemotactic factors following TBI (Karve et al., 2016, Mishra et al., 2016). These inflammation mediators participate in multiple pathways, which lead to energy depletion, protein aggregation and excitotoxicity. Eventually, TBI causes structural damage and neurological deficits, for instance, cognitive impairments and motor dysfunctions, directly related to cortical and hippocampal disruption with neuronal loss (Finnie, 2013, Xiong et al., 2009). Given the irreversibility of the primary injury, therapeutic targets for TBI focus on the secondary damages. Over the last three decades, more than 40 phase II and phase III clinical trials testing neuroprotective agents designed to enhance morphologic and functional recovery following TBI. However, the reported efficacy of numerous preclinical experimental therapies has not been translated into successful clinical trials. One possible reason is most of the researches focused exclusively on single therapy (Stein et al., 2015). The multifaceted and overlapping pathophysiological processes of TBI justifies the combination use of drugs for multiple molecular targets. In this regard, Chinese herbal medicine could be a new therapeutic strategy for TBI because it is usually a mixture of several constituents with diverse pharmacological functions which can simultaneously act at multiple sites (Yang et al., 2016).

Da Chuanxiong Formula (DCXF) is one of the most representative herbal formulas in traditional Chinese medicine (TCM). It was first recorded in “Xuan Ming Lun Fang” by Wan-Su Liu in Jin Dynasty. It contains dried rhizomes of Ligusticum chuanxiong Hort., umbelliferae (Chuanxiong Rhizoma, called as Chuanxiong in China, CR) and Gastrodia elata Bl., Orchidaceae (Gastrodiae Rhizoma, called as Tianma in China, GR) in the mass ratio of 4:1. This formula has been widely used to treat brain diseases like headache and migraine caused by blood stasis and wind pathogen. In DCXF, CR is employed as the monarch drug to promote “Qi” circulation in blood, expel wind and alleviate pain. The other herb, GR is used as the auxiliary drug to tranquilize the wind, terminate convulsion, calm the liver and suppress excessive “Yang”. Modern pharmacological investigations have shown that DCXF possesses therapeutic efficacies on various brain problems, including headache, migraine, vertigo, dementia, stroke and cerebral arteriosclerosis, due to its abilities to improve cerebral blood supply and blood vessel elasticity, decrease BBB disruption and edema formation, inhibit inflammation and nerve cell apoptosis, and reduce intracellular free calcium concentration (Wang et al., 2013). However, the therapeutic effect of DCXF on TBI has not been reported yet. Additionally, the guidelines of pharmacologic intervention for TBI is to developed pharmacologic solutions that can maximize efficacy and directly reduce cognitive and motor deficits after TBI (Elovic et al., 2008). According to the guidelines, the ultimate goal of any clinical study on TBI treatment is to address neurologic motor and cognitive problems and the experimental TBI researches also need to focus on the motor and cognitive function recovery following brain injury (Fujimoto et al., 2004). Thus, we investigated the neuroprotective efficacy of DCXF on cognitive and motor deficits using a well-established controlled cortical impact (CCI) model of TBI (Brody et al., 2007, Lam et al., 2016). In the present study, we demonstrate for the first time that DCXF attenuates both cognitive and motor impairments in the CCI-induced TBI rat model by Morris water maze test, acceleration rotarod motor test and catwalk quantitative gait analysis test, respectively, and its possible mechanisms that occur in the brain.