Peripheral nerves may be subjected to crush injuries in a variety of circumstances, including motor vehicle accidents, fractures, dislocations and natural disasters such as earthquakes [20
]. After injuries to peripheral nerves, axons and myelin sheaths distal to the lesion are degraded. The degenerative products are eliminated by the cooperative action of denervated Schwann cells and infiltrating macrophages. Wallerian degeneration serves to create a microenvironment favoring axonal regrowth. Schwann cells within the endoneurial tubes of the distal nerve dedifferentiate towards a non-myelinating proliferative phenotype that over-express growth factors, cell adhesion molecules and extracellular matrix to promote regeneration [1
In contrast to nerve transection injury, nerve crush injury causes less severity, because it leaves the basement membrane of Schwann cells surrounding the original nerve fibers intact and thus, despite the disrupted axon cylinder, enables Schwann cells to provide pathways to guide the regenerating axons [21
]. Although crushed peripheral nerves keep anatomical continuity, regenerate spontaneously and somehow reinnervate their target tissues, the longer it takes for the crushed nerve to reinnervate their target tissues, the greater the chance of permanent denervation atrophy of the target tissues. Therefore, accelerated nerve regeneration is crucial to obtain satisfactory functional outcomes [22
Functional deterioration following crush injury is not only related to the impact of the crush itself, but also includes other important components such as ischemia of the limb. Studies on crush injury models in peripheral nerves have shown better functional recovery when therapies were directed against ischemia-reperfusion injury by using antioxidants, lipid peroxidation inhibitors and anti-inflammatory agents [23
]. With this in mind, the present study evaluated the nerve regeneration activity of H. erinaceus
which possesses antioxidant activity as an alternative herbal pharmacotherapy for peripheral nerve repair [24
The functional recovery as assessed by PFI scores in couple with histological examination of regenerated nerves and target EDL muscle revealed that aqueous extract promoted peripheral nerve regeneration. It was also noted that the neuroprotective effect of aqueous extract was approaching to those elicited by mecobalamin.
The hind limb function served by the sciatic nerve and its branches in the rat can be quantitatively, reliably and easily assessed by gait analysis through foot prints [18
]. The clinically relevant outcome after peripheral nerve injury is the functional recovery of end organ or muscle which is the ultimate test of nerve regeneration. If physical contact is restored between a motor neuron and its muscle, function is usually reestablished [25
]. Carlton and Goldberg introduced the PFI [26
], which was later modified by Bain et al. [18
]. PFI method is suitable for evaluation of a complete lesion of the peroneal nerve which produces a short and narrow foot print due to the lack of dorsal flexion of the ankle and extension of the toes [25
]. Although each group demonstrated improvement during the post-surgery period, complete functional recovery was not attained in negative control group until 14 days after injury. Aqueous extract or mecobalamin provides a quicker functional recovery by 4–7 days earlier than negative control group. In spite of this, there was no significant difference in PFI values among all treated groups, showing that different doses of aqueous extract might not have significant effect on functional recovery.
Numerous neurofilament immunopositive axons were seen in peroneal nerve sections from operated limb of aqueous extract and mecobalamin groups. The density of neurofilament immunopositive areas in negative control group was greatly decreased. In fact, nearly all rats in this group sustained at least moderate axonal loss after 14 days of injury.
During the early stages of mammalian ontogeny, muscle fibers are innervated by more than one axon [27
]. Polyneuronal innervation is then replaced by mononeuronal innervation in the course of development by mechanical activity of the muscle fiber [28
], so that in the rat most muscle fibers are contacted by only one axon by the end of the second week of life [17
]. Polyneuronal innervation is rare in normal skeletal muscle. However, it affects a high number of muscle fibers and motor neurons during the first stages after nerve section and regeneration [29
]. As the rat recovers its motor function, withdrawal of polyneuron will lead to mononeuronal innervation. If we take the percentage of polyneuronal innervation as a measure of motor endplate maturation, it can be concluded that the rate of maturation after peroneal nerve crush is accelerated in mecobalamin group as compared to aqueous extract groups. Functional connections of severed axons to regenerate and facilitate adequate target reinnervation of EDL muscle was speeded up in treated groups.
Natural products have been traditionally accepted as remedies due to popular belief that they present minor adverse effects [30
]. Mushrooms have always been prepared for medicinal use by hot water extraction in traditional Chinese medicine. The extraction was with hot water as in brewing of teas or decoctions. This method is used to prepare extracts of commonly used mushrooms including shiitake, maitake, cordyceps, coriolus and reishi. Hot water extraction has been used for all the well-known products such as lentinan and LEM from shiitake, Maitake d
-fraction and MaitakeGold 404 from maitake, and PSP from Coriolus versicolor
Aqueous extract is comprised of polysaccharides including acidic heteroglycans, β
-glucans and glucuronoxylomannan. Polysaccharides perform numerous functions in various cell types such as neuritogenesis, peripheral nerve regeneration and muscle reinnervation following a sciatic nerve lesion [32
]. Moreover, from a toxicological point of view, water is safer than organic solvents such as acetone, chloroform and methanol.
Sugar and polysaccharide contents in Malaysian grown H. erinaceus
have been quantified by Choong et al. [33
]. The high-performance liquid chromatography (HPLC) analysis of the hot water crude extract showed arabinose as the major component with minor components of glucose, rhamnose, deoxyribose and galactose. The presence of free arabinose in hot water crude extract has not been previously reported. Their finding showed that the fruiting bodies of locally grown H. erinaceus
contained polysaccharides components which were mainly arabinose and not glucose as mentioned in some studies from China [34
The dose of 10
mL or 20
body weight per day is based on repeated trials or historical practices. In a study performed in Korea, single- and repeated-dose toxicity studies of Erinacol, the water extract of H. erinaceus
cultivated with Artemisia iwayomogi have been evaluated according to “Guidelines for Toxicity Tests of Drugs and Related Materials" of Korea Food and Drug Administration using Sprague-Dawley rats [35
]. Erinacol up to the limited dose of 5000
mg extract kg−1
neither induced death, clinical signs and necropsy findings, nor affected body weight gain and organ weights. The yield of 10
g of fresh fruiting bodies after boiling with 10
mL of distilled water was about 10
mL of aqueous extract. Ten grams of fresh fruiting bodies is equivalent to 1
g of dried powder. Therefore 10 or 20
body weight per day is comparable to 1000 or 2000
mg of dried powder per kg body weight per day. In the present study, the doses of aqueous extract have not shown any signs of immediate danger and exhibited no toxicity in rats.
With regard to its assumed mechanism of action, the improved regeneration observed after aqueous extract treatment may be related either to a direct neurotrophic factors-like activity or to the promotion of the effects of nerve-derived neurotrophic factor. Neurotrophic factors or regeneration-promoting factors have been suggested to play an essential role in the outcome of degeneration and regeneration processes in the peripheral nervous system, both to ensure proper innervation of the target tissues and to improve remyelination [36
]. It may also affect the adherence of platelets and macrophages and the release of cytokines such as tumor necrosis factor (TNF), leading to decreased permeability and tissue edema and better capillary perfusion ().
Hypothetical diagram showing the possible effects of aqueous extract of H. erinaceus fresh fruiting bodies in promoting peripheral nerve regeneration following crush injury.
By taking natural products into consideration, the repair effect of the traditional Chinese medicinal herb, Achyranthes bidentata
Blume root aqueous extract on regeneration of the crushed rabbit common peroneal nerve was studied by Ding et al. [37
] and crushed mouse sciatic nerve by Yuan et al. [22
] using a combination of electrophysiological assessment and histological investigation. The root extract could accelerate peripheral nerve regeneration in a dose-dependent manner. Lumbricus and Radix Hedysari aqueous extracts have been shown to produce a positive effect on the motor function recovery and conductivity recovery following sciatic nerve clamping injury by increasing the total number of regenerated myelinated nerve fibers in adult rats [38
]. Lumbricus is a cold and slightly salty traditional Chinese medicine that derived from the abdomen of earthworm while Radix Hedysari is the dry root of Hedysarum polybotrys
Hand.-Mazz. Jiang et al. also [40
] demonstrated that chitooligosaccharides, the biodegradation product of chitosan, promoted peripheral nerve regeneration with the desired functional recovery in the rat sciatic nerve crush injury model.
From this study, we have shown that aqueous extract of H. erinaceus
fresh fruiting bodies, administered at a non-toxic dose of 10 or 20
body weight per day increased the rate of recovery after peripheral nerve injury. However, treatment with different doses of aqueous extract did not have a statistical significant difference in recovery as assessed in behavioral experiment and histological examinations. Therefore, low dose of 10
body weight per day would be sufficient in facilitating functional recovery after peripheral nerve injury. Patients who receive H. erinaceus
may experience a more expeditious improvement in the quality of life and a more complete functional recovery after injury. Moreover, by taking mecobalamin for the treatment of nerve injury gives rise to side effects such as gastrointestinal and dermatological problems [41
]. Future research models will focus on direct effect and mechanism of action of H. erinaceus
on peripheral nerve regeneration and refining strategies to enhance regeneration.