Neuroprotective effects of Tilia americana var. mexicana on damage induced by cerebral ischaemia in mice

Abstract Tilia americana var. mexicana (T. americana) is a plant widely used in Mexico for its medicinal properties on the central nervous system. In the present study, we designed a protocol to investigate the neuroprotective effects of non-polar and polar extracts of T. americana on damage induced by cerebral ischaemia in mice. Vehicle or extracts were administered immediately after ischaemia. Functional neurological deficit, survival percentage and infarct area were determined in each experimental group. Results showed that groups treated with non-polar or polar extracts of T. americana had increased survival rate, improved neurological deficits and diminished the infarct area in relation to the ischaemic group. In conclusion, this study confirms the neuroprotective activity of T. americana, suggests a possible synergism between non-polar and polar constituents and supports its potential as a useful aid in the clinical management of stroke.


Introduction
Stroke is considered the first cause of disability and the third cause of death in developed countries. Ischaemic strokes are characterised by a reduction in blood flow to brain tissue, resulting in a corresponding loss of neurological function or death (Moretti et al. 2014). The pathophysiology of stroke is complex and involves numerous processes that include ABSTRACT Tilia americana var. mexicana (T. americana) is a plant widely used in Mexico for its medicinal properties on the central nervous system. In the present study, we designed a protocol to investigate the neuroprotective effects of non-polar and polar extracts of T. americana on damage induced by cerebral ischaemia in mice. Vehicle or extracts were administered immediately after ischaemia. Functional neurological deficit, survival percentage and infarct area were determined in each experimental group. Results showed that groups treated with non-polar or polar extracts of T. americana had increased survival rate, improved neurological deficits and diminished the infarct area in relation to the ischaemic group. In conclusion, this study confirms the neuroprotective activity of T. americana, suggests a possible synergism between non-polar and polar constituents and supports its potential as a useful aid in the clinical management of stroke. depletion of oxygen and ATp by the interruption of blood flow, excitotoxicity, inflammation, oxidative damage and apoptosis of brain tissue; this explains why therapies currently available for its treatment are practically ineffective. The only approved drug in the United States for the treatment of acute ischaemic stroke is recombinant tissue plasminogen activator; however, its use is limited by its narrow therapeutic window. Therefore, exploration of new therapeutic alternatives for its treatment continues to be an important issue (Kaur et al. 2013). Medicinal plants could constitute one of such alternatives (Ghosh et al. 2014). In recent preclinical studies, some extracts of Knema laurina showed neuroprotective activity against cell death induced by oxidative stress in human neuroblastoma cells (Ismail et al. 2015); and, Bacopa monnieri (Brahmi), a ayurvedic medicinal herb, showed neuroprotective activity in neurotoxicity induced by methyl mercury (Ayyathan et al. 2015).
Tilia americana var. mexicana (Schltdl.) Hardin (Malvaceae) (T. americana) is a plant growing in Mexico that has been used in traditional medicine for its anxiolytic properties. Its sedative, tranquiliser, analgesic and anti-inflammatory effects have been confirmed in a scientific study (Martínez et al. 2009). Recently, we reported an in vitro neuroprotective effect of extracts of this plant on neuronal damage induced by intestinal ischaemia in situ in the myenteric neurons (Angeles-López et al. 2013). The present study more directly evaluates the neuroprotective effect of polar and non-polar extracts of T. americana in an in vivo model of cerebral ischaemia in mice.

Neurological impairment and mortality induced by cerebral ischaemia
The pathological manifestations in stroke are diverse and depend on the severity, duration and localisation of the ischaemic damage. Many studies have demonstrated that the increase in oxidative stress induced during ischaemia contributes to neuronal damage (Kaur et al. 2013). In this study, cerebral ischaemia was induced in mice by sequential carotid artery sectioning (SCAS) (Rodriguez et al. 2000), which produces diverse neurological alterations expressed by the neurological disability status scale as described in supplementary material. In the ischaemic group, the mean of neurological scores were 5.6 ± 0.6, 7.5 ± 0.9 and 8.4 ± 0.6 at 24, 48 and 72 h, respectively. Such alterations were not modified by the vehicle (5% Tween 80 in saline solution, ISCH-VEH); but they were diminished by aqueous (ISCH-AQUO) or hexane (ISCH-HEX) extracts of T. americana, which showed neurological scores of 4.0 ± 0.5, 4.8 ± 0.5 and 5.9 ± 0.7; and, 3.7 ± 0.5, 4.1 ± 0.5 and 5.0 ± 0.5, respectively, at the evaluated times ( Figure S1a). No neurological alterations were observed in animals without ischaemia (SHAM group). Cerebral ischaemia also caused death in 15% of animals of the ISCH-VEH group one hour after the second surgery; this increased progressively to 55% at 24 and 48 h, and 88% at 72 h. Treatment with aqueous or hexane extracts diminished ischaemia-induced mortality to 37, 49 and 69% (ISCH-AQUO); and, 31, 47 and 68% (ISCH-HEX) at 24, 48 and 72 h, respectively. In the SHAM group no deaths were recorded. In this study, deaths induced by ischaemia were expressed as per cent of survival and were illustrated by Kaplan-Meier survival curves ( Figure S1b). Analysis by the log-rank test showed greater per cent survival in ischaemic groups treated with either extracts of T. americana in comparison with the ISCH-VEH group (p < 0.05). These results demonstrate that acute administration of aqueous or hexane extracts of T. americana confers partial neuroprotection against damage induced by cerebral ischaemia in mice. Some of the chemical constituents present in this species could be responsible for the neuroprotective effect observed in this study. Recently, the capacity to trap multiple reactive species by flavonoids present in extracts of T. americana was reported suggesting their antioxidant activity (Cárdenas- Rodriguez et al. 2014). Flavonoids are the largest group of polyphenols whose capacity to act as antioxidants is related to the abundant phenolic hydroxyl groups present in their molecular structure (Galleano et al. 2010). Using a high-performance liquid chromatography and mass spectrometry technique (HpLC-MS), several flavonoids derived from kaempferol (2.2 ± 0.18 mg/g) and quercerin (16.8 ± 2.8 mg/g), such as the glycosides rutin, astragalin, tiliroside, kaempferitrin and isoquercitrin, have been isolated from this species (Aguirre-Hernández et al. 2010). In other studies, flavonoids have been implicated in the neuroprotective effects of Rosa laevigata and Phoenix dactyliferia against damage induced by cerebral ischaemia (pujari et al. 2013; zhang et al. 2013). In addition, quercetin and tiliroside have shown activity as scavengers of free radicals (Sala et al. 2003), and rutin attenuated neural apoptosis by increasing endogenous antioxidant enzymatic activities in rats subjected to cerebral ischaemia (Khan et al. 2009).
On the other hand, there are also non-polar natural antioxidants (terpenes and phytosterols) which act as hydrogen donors, reducing the lipidic peroxidation or inflammation (Takahashi et al. 2003). In the hexane extract of T. americana, β-sitosterol (a phytosterol), heptacosane (a hydrocarbon) and palmitic acid (a fatty acid) have been identified through thin-layer chromatography and gas chromatography/mass spectral analysis (Aguirre-Hernández et al. 2007). preclinical studies have showed that β-sitosterol (isolated from Aloe vera) induced neuroprotection in a model of ischaemia-reperfusion in Mongolian gerbils, an effect attributed to its angiogenic activity and its capacity to prevent lipid peroxidation (Baskar et al. 2012); on the other hand, palmitic acid did not protect mice after focal brain ischaemia-reperfusion (Heurteaux et al. 2006). No information was found on the effect of heptacosane in models of ischaemia.
In spite of evidence of neuroprotection for some of the identified constituents of both non-polar (hexane) and polar (aqueous) extracts of T. americana, the precise mechanism involved in the effects shown in this study is unknown; but it is possible that some of them may be acting at the same time through several pathways. Thus, the combination of hydrophilic and lipophilic natural antioxidants in medicinal plants may exert synergistic effects to promote a preventive strategy against ischaemia.

Determination of infarct area
In relation to the size of the infarct induced by cerebral ischaemia in the SCAS model, this was not altered by vehicle (ISCH-VEH) (data not shown), but was diminished after treatment with either extracts of T. americana . This reduction was significant in the ISCH-AQUO group at 48 and 72 h; while, in the ISCH-HEX group, only reached statistical difference at 72 h. No infarct area was detected in the SHAM animals ( Figure S2a, b). According to Rodriguez et al. (2000), cerebral cortex and striatum in both hemispheres are brain areas susceptible to damage induced by ischaemia in this model ( Figure S2b). It is known that the cerebral cortex is involved in processing and interpreting sensory information, planning and organising tasks and controlling motor activities, while the stratium receives input from the cerebral cortex and constitutes the primary input to the basal ganglia system (von Nicolai et al. 2014). Thus, integrity of the corticostratial structures is essential for intact motor functioning. The damage induced by ischaemia in these cerebral areas in the SCAS model explains the neurological impairment observed in this study; and, the reduction in these areas in animals treated with polar (ISCH-AQUO) and non-polar (ISCH-HEX) extracts of T. americana may explain the decreased neurological alterations observed in these groups. In general, infarct size is one of the most commonly used endpoints to assess the efficacy of potential therapies, although most evidences indicate that the capacity to reduce infarct size in laboratory animals does not always correlate with neuroprotective effects in clinical studies (Rodriguez et al. 2000). Since most agents that reduce infarct size in laboratory animals have failed in clinical practice, an accurate assessment of the neurological status of ischaemic animals in preclinical studies is essential to validate the efficacy of a given treatment. Thus, some authors consider that estimation of functional capacity constitutes a reliable measure to determine the course of impairment and the consequences of pharmacological manipulations.

Conclusions
In conclusion, these results support the use of this plant in folk medicine and suggest its utility for stroke prevention. Additional studies to identify the metabolite or metabolites responsible for the neuroprotective effect and their mechanism of action seem warranted, as are clinical trials to confirm the efficacy of the T. americana in the prevention of stroke.

Supplementary material
Experimental details are available in supplementary material, alongside Figures S1 and S2.

Disclosure statement
No potential conflict of interest was reported by the authors.