Saffron extract and crocin exert anti-inflammatory and anti-oxidative effects in a repetitive mild traumatic brain injury mouse model

Abstract

Saffron Crocus sativus L. (C. sativus) is a flower from the iridaceous family. Crocin, saffron's major constituent, and saffron have anti-oxidative and anti-inflammatory activities. In this work, the neuroprotective effects of saffron and crocin are being investigated in a repetitive mild traumatic brain injury (rmTBI) mouse model. A weight drop model setup was employed to induce mild brain injury in male albino BABL/c mice weighing 30-40 g. Saffron (50 mg/kg) and crocin (30 mg/kg) were administrated intraperitoneally 30 min before mTBI induction. Behavioral tests were conducted to assess behavioral deficits including the modified neurological severity score (NSS), Morris water maze (MWM), pole climb test, rotarod test, and adhesive test. The levels of TNF alpha (TNF-α), interferon-gamma (IFN-γ), myeloperoxidase activity (MPO), malonaldehyde (MDA), and reduced glutathione (GSH) were measured. Histological analysis of different brain parts was performed. Both saffron and crocin demonstrated marked improved neurological, cognitive, motor, and sensorimotor functions. Besides, both compounds significantly reduced the oxidative stress and inflammatory processes. No abnormal histological features were observed in any of the injured groups. Saffron extract and crocin provide a neuroprotective effect in a mouse model of rmTBI by decreasing oxidative stress, inflammatory responses, and behavioral deficits.

Figure 1

The weight difference between day 0 and day 14. Data are mean ± SEM of 25 mice per group. TBI group showed a negative difference (p < 0.0001). Both saffron TBI group and crocin TBI showed a positive difference (p < 0.0021).

Figure 2

Neurological severity score (NSS) for mice in all experimental groups. Data obtained showed that the TBI group presented higher NSS than the sham group. Saffron and crocin treatment reduced the NSS score. Data are mean ± SEM of eight mice per group.

Figure 3

Morris water maze results of all experimental groups following TBI. Short-term defects in spatial learning were detected in the TBI group. Saffron and crocin enhanced the induced cognitive deficits and both groups showed no difference in their latency to reach the platform a: sham, b: saffron sham, c: crocin sham, d: TBI, e: saffron TBI, f: crocin TBI. Data are mean of eight mice per group. (*) and (**) represent p < 0.03 and < 0.0021 respectively.

Figure 4

Pole climb, rotarod, and adhesive tests results. (a, b) show t1/2 and t values respectively in pole climb test results. Data is mean ± SEM of eight mice per group. (****) represents p < 0.0001. (c) The latency to fall from the rotarod in (s). The latency was analyzed for all experimental groups. Data is mean ± SD of eight mice per group. (**) and (****) represent p < 0.0021 and < 0.0001 respectively. (d) The latency to remove the tape in adhesive test for all experimental groups. Data are mean ± SEM of eight animals in each group. (****) represents p < 0.0001.

Figure 5

H&E stain of brain tissue of BALB/c mice in all experimental groups. Sections (a–f) show parts of the cortex of the following groups sham, saffron sham, crocin sham, TBI, saffron TBI, and crocin TBI respectively (4×). Sections (g–l) show parts of the denta gyrus of the following groups, sham, saffron sham, crocin sham, TBI, saffron TBI, and crocin TBI respectively (10×). Sections (m–r) show parts of the cerebellum of the following groups, sham, saffron sham, crocin sham, TBI, saffron TBI, and crocin TBI respectively (20×). Sections (s–x) show parts of the corpus collosum of the following groups, sham, saffron sham, crocin sham, TBI, saffron TBI, and crocin TBI respectively (20×). All sections appear without any neuronal loss and without any axonal spheroids among all groups. No evidence was shown for tissue loss, contusions, edema, or intraparenchymal hemorrhages in sections of all groups. All sections shown were obtained after 7 days from the final injury.

Figure 5

H&E stain of brain tissue of BALB/c mice in all experimental groups. Sections (a–f) show parts of the cortex of the following groups sham, saffron sham, crocin sham, TBI, saffron TBI, and crocin TBI respectively (4×). Sections (g–l) show parts of the denta gyrus of the following groups, sham, saffron sham, crocin sham, TBI, saffron TBI, and crocin TBI respectively (10×). Sections (m–r) show parts of the cerebellum of the following groups, sham, saffron sham, crocin sham, TBI, saffron TBI, and crocin TBI respectively (20×). Sections (s–x) show parts of the corpus collosum of the following groups, sham, saffron sham, crocin sham, TBI, saffron TBI, and crocin TBI respectively (20×). All sections appear without any neuronal loss and without any axonal spheroids among all groups. No evidence was shown for tissue loss, contusions, edema, or intraparenchymal hemorrhages in sections of all groups. All sections shown were obtained after 7 days from the final injury.

Figure 6

Effect of saffron extract and crocin on IFN-γ and TNF-α Levels in cortex homogenates. (a) IFN-γ levels in all groups. (b) The concentrations of TNF-α. Data are represented as means of three determinations ± SEM. (****) corresponds to p-value < 0.0001.

Figure 7

Effect of saffron extract and crocin on MPO activity, MDA content, and GSH levels. (a) The significant increase in activity of MPO in TBI, saffron TBI and crocin TBI groups and shows the relative decline in MPO activity in saffron TBI and crocin TBI groups compared to the TBI group. MDA content is expressed in (b). The level of MDA is significantly high in the TBI, saffron TBI, and crocin TBI groups. (c) GSH levels in the experimental groups. TBI caused a significant decrease in GSH levels while saffron and crocin treatment restored normal GSH levels. Both treatments were significantly effective in reducing MDA levels. Data are represented as means of three determinations ± SD. (****) corresponds to p-value < 0.0001.

Figure 8

Scheme of TBI apparatus used to induce brain trauma.