Introduction

Neuronal degeneration, i.e., neuronal cell death, underlies the pathology and malfunction of many different neurological diseases occurring in both animals and human beings. Progressive and selective neuronal cell death in the central nervous system (CNS) and/or the peripheral nerve network has been profoundly implicated in the pathogenesis of neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease, Huntington’s disease, Lou Gehrig’s disease or amyotrophic lateral sclerosis, multiple sclerosis, epilepsy, stroke, traumatic injury, age-related macular degeneration, glaucoma, prion diseases, infections, and so on [1, 2]. Evidence is rapidly accumulating to suggest that selective neuronal cell death through necrosis and/or apoptosis mechanisms contributes significantly to the functional anomalies of specific neurologic disorders [1]. Changes of genetic, epigenetic, metabolic, and environmental factors might directly or indirectly cause (1) massive DNA damage, (2) dysfunction of the ubiquitin-proteasomal system, (3) disruption of the axonal transport machinery, (4) abnormalities of mitochondrial structure and function, (4) disturbance of intracellular ionic homeostasis (particularly Ca2+ and Zn2+), and (5) accumulation of reactive oxygen species (ROS) in the neuronal cells. Accumulating intracellular stress subsequently results in (1) loss of spines and synapses, (2) fragmentation of neuronal processes and extended neuritic degeneration following demyelination, (3) global neuronal cell death following activation of signal transduction cascade for programmed cell death, (4) anomalies of microvasculature, and (5) provocated neuroinflammatory response leading to the destructive pathogenic changes (see several representative reviews) [1–4]. Accordingly, the main aim of neural protection in neurodegenerative disorders is to retard progression by blocking the mechanisms that lead to neuronal cell death as well as associated neuroinflammatory events. Therefore, considerable efforts have been made in recent decades to discover new potential therapeutic compounds that can help to prevent the onset or to slow down the progression of such diseases. Equal attempts are also made to improve the therapeutic efficacy of known medications through chemical modification. Given the evident advantages of being stable in the ambient environment, being permeable to the blood–brain and/or blood–eye barriers, and being convenient for administration (Fig. 1), naturopathic compounds have growingly become groups of the best therapeutic candidates for neural protection from the sensory system including retina to the central nervous system (brain). Table 1 summarizes a group of selected herbal extracts that have demonstrated significant neuroprotective efficacy both in vivo and in vitro. Taking tetramethylpyrazine (TMP) as an example, the neuroprotective efficacy and related issues are discussed here.

Fig. 1
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The conventional route of most natural compounds from herbs to target organs. In the traditional Chinese medicine, the herbal therapeutic ingredients are extracted and prepared as herbal tea or soup with water through regular cooking and taken orally. The effective ingredients are absorbed by the gastrointestinal tract and successfully pass through the blood–brain and/or blood–eye barriers to reach the targets, brain and/or retinas (graphic clips were taken from the Internet free sources)

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Table 1 List of selected naturopathic compounds that have demonstrated neuroprotective efficacy in both retina and brain
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Tetramethylpyrazine, an herbal extract showing multiple protective effects on cells and benefits on physiological function

As listed in Table 1, TMP, also designated as ligustrazine, is an alkaloid extracted from the Chinese herbal medicine, Ligusticum wallichii Franchat (chuanxiong) [5]. For hundreds of years, chuanxiong has been used as a traditional Chinese medicine for heart, kidney, and brain diseases [6, 7]. Experimental studies demonstrated that TMP treatments significantly improved cardiac and cerebral blood flow and elevated blood reperfusion as shown in the nail microcirculation [8, 9]. In an ex vivo study, a semi-synthetic form of TMP monomer induced a dose-dependent relaxation of human pulmonary and bronchial arteries [10]. TMP also exhibited a calcium antagonist role in vascular tissues [11]; functioned as a ROS scavenger to deactivate cytotoxic ROS such as superoxide anion (O2−), hydroxyl (OH−), and lipid peroxyl (LOO−) free radicals [12, 13]; and inhibited inflammatory events in vivo possibly through modulating secretion of specific cytokines and nitric oxide-related pathways [14–16].

About two decades ago, a study briefly reported that TMP alleviated ischemic retinal degeneration in vivo [17]. Recently, a different group of researchers demonstrated that systemic injection of TMP significantly protected retinal photoreceptor from loss induced by N-methyl-N-nitrosourea in rats [18]. Further, we demonstrated that TMP efficiently enhanced in vitro survival of cultured rat retinal cells and significantly attenuated cell damage in these cells exposed to hydrogen peroxide [19].

In the CNS, TMP significantly suppressed oxidative stress and attenuated neuronal cell death in neuronal cultures following iron-mediated oxidative damage and glutamate-mediated excitotoxicity [20–22]. Systemic administration of TMP protected neuronal cells against ischemic or traumatic brain or spinal cord injury and promoted functional recovery in rodents and rabbits [23, 24]. Interestingly, systemic administration of TMP also attenuated impairment of learning and memory performance in rodents following d-galactose- or ischemia-induced brain injury [25, 26]. The potential therapeutic efficacy of TMP for AD is further supported by our recent observations about significant improvement of cognitive function as well as cerebral amyloid pathology in the demented Alzheimer’s triple transgenic (3xTg-AD) mice (Tan et al., unpublished observations). These findings suggest that TMP has potent neuroprotective efficacy.

TMP protects neuronal degeneration in rat brain against excitotoxicity—an experimental study

Systemic administration of excitotoxin, kainic acid (KA), a glutamate analog, causes striking neuronal cell death in rat brain following prolonged seizures. To examine further whether TMP protects brain cells from KA-induced neuronal degeneration, which is one of the most common animal models for excitotoxic neuronal cell death that is apparently involved in the pathogenesis of multiple neurodegenerative disorders [27–29], 1-month-old male Sprague–Dawley rats were housed and treated according to the National Institutes of Health guidelines for the care and use of laboratory animals and a protocol approved by the UCI Institutional Animal Care and Use Committee. One hour after the onset of seizures following subcutaneous (s.c.) injection of KA (10.5 mg/kg), rats received TMP (50 mg/kg, s.c.) or equal volume of vehicle. Untreated animals were used as blank controls. All the animals (N = 6 each group) were decapitated 24 h after TMP or vehicle injection under CO2 gas-induced deep anesthesia. The brain was rapidly harvested, frozen, and cryosectioned in the coronal plane at 10 µm. Adjacent sections from each brain were stained with hematoxylin–eosin (H & E) and in situ cell death assay kit for terminal deoxytransferase-mediated dUTP nick end labeling (TUNEL; Roche, Indianapolis, IN, USA), respectively, as described in our previous work [30, 31].

Systemic administration of KA resulted in a well-described pattern of behavioral seizures including wet dog shake at the beginning stage and progressed to tonic–clonic activity [27]. Seizures typically occurred intermittently during the first 6–8 h and yielded about 10% or less mortality. As observed in this study, TMP treatments did not alter the pattern and the severity of the seizures. Brain sections from the animals that received “KA + vehicle” demonstrated robust neuronal degeneration in KA-vulnerable regions in brain as revealed by both H & E staining and TUNEL labeling (Fig. 2a (f–j)) whereas the controls showed no eosinophilic or TUNEL-positive damaged cells (Fig. 2a (a–e)). In contrast, given 50 mg/kg TMP following the onset of seizures, animals showed markedly fewer damaged cells in the corresponding regions in the brain (Fig. 2a (k–o)). Quantification of TUNEL-positive cells conducted as described in our previous work [32, 33] revealed a statistically significant difference between “KA + vehicle” and “KA + TMP”, suggesting remarkable neuroprotective efficacy of TMP in the CNS under excitotoxic attack.

Fig. 2
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Systemic administration of TMP attenuates neuronal degeneration in rat brains following kainate-induced seizures. a Both hematoxylin–eosin (H & E) and TUNEL staining demonstrate massive neuronal damage shown as pink in H & E staining (f, g) and green in TUNEL (h, i, and j) in both hippocampus (f, g, h, and i) and piriform cortex (j) regions. In contrast, the animals received TMP only exhibited a few sporadic eosinophilic (l) and TUNEL-positive cells in the corresponding regions (m, n, and o). b Quantification of TUNEL-positive cells according to our previous methods reveals a significant decrease in the number of damaged neurons in the hippocampal regions with TMP treatment. Scale bar = 100 µm in b, d, e, g, i, j, l, n, and o; and 400 µm in a, c, f, h, k, and m

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In cultured neural cells, TMP treatments significantly reduced generation of lipid peroxidation products, malondialdehyde, induced by hydrogen peroxide [19, 20]. The observations are also in agreement with an increase in abundance of glutathione in 3xTg-AD mouse brains following TMP treatments (data not shown). In addition, the benefits of TMP treatments preserved abundance of MAP2 and rattin, two molecules that play important roles in cell growth and function [19, 34, 35]. Taken these together, TMP may target multiple levels and cell signal transduction pathways to contribute to the survival of neural cells and the normal function of the nervous system.

How far is TMP from the next phase for clinical applications?

As a naturopathic compound isolated from a Chinese herbal medicine, TMP has been the subject of many pharmacological and toxicological studies. The solubility of purified TMP in crystal form is relatively low in neutral aqueous solution (~10 µg/ml) and dramatically increases in an acidic environment (>40 mg/ml, pH < 4), in which TMP is stable and active [36]. Preclinical assessment of the distribution, metabolism, excretion, and toxicity (ADMET) of TMP has been performed in animals and in vitro since more than 20 years ago [37–41]. Without any surprise, pharmacokinetic studies demonstrated that TMP was efficiently permeable to the blood–brain barrier in multiple animal models [39, 40]. Toxicity assays revealed a very low level of toxicity in animals with an oral LD50 of about 1,910 mg/kg in rats and 1,436 mg/kg in mice [42, 43]. Importantly, practitioners of traditional Chinese medicine have continued to use TMP as a treatment for inflammatory or degenerative diseases, usually in combination with other medications [6, 8, 44–46]. In this regard, both TMP tablets and TMP–HCl injection solution are prescription-available for a treatment of cardiovascular diseases in China [47, 48]. Given the ADMET information that is already available on TMP, it would not take much preclinical data to justify bringing TMP to clinical trials in humans. This proof-of-concept proposal will provide sufficient preclinical evidence to justify studying TMP in patients with neurodegenerative disorders in a double-blind placebo-controlled manner. Nevertheless, our understanding of the molecular basis for TMP-mediated pharmacological actions remains limiting. Similarly, there is a lack of well-designed preclinical studies of TMP efficacy for neurological disorders. Such studies are critical prior to moving TMP to studies in humans.

Summary

In addition to TMP, there are groups of naturopathic compounds that have been purified from related herbal medicines and identified as efficient neuroprotective ingredients as mentioned. Resembling TMP, many of these compounds have demonstrated remarkable neuroprotective efficacy in experimental studies conducted in cell cultures and/or live animals. Some of them are also used in clinics as treatment for specific neurological disorders. Therefore, once further studies are warranted to decipher the molecular basis of related pharmacological efficacy, many of such naturopathic molecules would move to clinical assessments for neural protection in humans.