Sea urchin (Diadema savignyi) extract as a novel protective agent against cisplatin induced neurotoxicity in rats

Abstract

Neurotoxicity is a severe side effect of platinum compounds used for cancer chemotherapy such as Cisplatin. This neurotoxicity leads to severe cognitive and nervous dysfunction, therefore, limiting the dose of Cisplatin and compromising the treatment protocol.The present study investigates the neuroprotective effect of Sea Urchins which is a marine animal known for its rich bioactive compounds. Male Sprague Dawley rats received Cisplatin (2 mg/kg body weight) for 4 weeks, two times per week, followed by Sea Urchin extracts (50 and 100 mg/kg body weight) twice weekly for 4 weeks.Results show that rats treated with Urchin's extracts showed a significant improvement in the thermal (heat and cold) sensitivity compared to untreated rats. Liver enzymes Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) and Urea levels were also significantly decreased back to normal following treatment with sea urchin extracts. Brain tissue oxidative stress marker Nitric oxide (NO) and lipid peroxidation marker Malondialdehyde (MDA) increased significantly in the cisplatin-treated rats while the reduced glutathione levels (GSH) and catalase activity (CAT) showed a significant decrease. Treatment with sea Urchin extracts reversed these changes.Histological and immunohistochemical examination of the cerebral cortex reveled degenerative changes such as karyopyknosis and shrunken necrotic ghost like neurons in the cisplatin treated groups. There was also strong positive Glial fibrillary acidic protein (GFAP) reactivity and a negative B-cell leukemia/lymphoma 2 protein (Bcl2) reaction in most apparent neurons, indicating strong apoptotic changes. Treatment with Urchin extracts reversed these changes. Quantification of cerebral cortex neurons also revealed the strong effect of the extracts. Cisplatin treated groups showed 3708 cells/ mm3 compared to 8091 cells/mm³ in the normal rats. Extract treatment increased the neuronal numbers to almost normal levels. Quantification of the Immuno-histochemical expression of GFAP showed an increase by 10-folds after cisplatin administration. A remarkable decline from the cisplatin group was seen in the extract treated groups.In Conclusion, Sea Urchins extracts possess a strong neuroprotective activity and could provide a novel therapeutic method to prevent Cisplatin-induced neurotoxicity.

Keywords: Bcl2; Cisplatin; Cisplatin induced toxicity; Diadema Savignyi; GFAP; Neurotoxicity; Oxidative stress; Sea urchins.

Fig. 1

Behavioral Assessment. Cisplatin administration significantly decreased cold withdrawal latency for cold (a) from about 30 sec to about 5 seconds (**** p-value < 0.0001). Treatment with shell and spine extracts increased the latency back to control levels, except for the SP-100 group which was still below the control levels. (*p-value < 0.05, n = 6). Heat withdrawal latency decreased from 15 sec to about 5 seconds (**** p-value < 0.0001) following Cisplatin administration. Treatment with shell and spine extracts increased the latency back to control levels, except for the SH-100 group which was longer than control levels. (*p-value < 0.05, n = 6)

Fig. 2

Kidney and liver function tests. Cisplatin administration increased ALT, AST, Urea and Creatinine (**** p-value < 0.0001). Treatment with sea Urchin extracts reduced Creatinine levels below those of the cisplatin treated group (#### p-value < 0.0001). There was no significant difference between the different treated groups and control. On the other hand, Urea levels were significantly lower than normal control and cisplatin group (*p-value < 0.05, #### p-value < 0.0001). ALT and AST were also reduced back to normal levels. The AST levels in the SH and SP 50 mg treatment groups were significantly higher than normal (n = 5, *p-value < 0.05)

Fig. 3

Hematological parameters. Cisplatin administration caused significant changes in hematological parameters Hemoglobin, red blood cell (RBC), white blood cells (WBC), and platelets. Treatment with both spine and Shell extracts reversed these changes. The shell 100 mg treatment group showed higher levels of RBCs compared to normal. In contrast, the spine 100 mg treatment caused a reduction in WBCs below normal levels. n = 6, *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; **** p-value < 0.0001. Platelets, RBCs, WBCs, and Hemoglobin levels were significantly different from those of the cisplatin treated group (#### p-value < 0.0001)

Fig. 4

Oxidative Stress Markers. Cisplatin administration caused a significant increase in Nitrous oxide (NO) levels and Malondialdehyde (MDA) levels in the brain tissue. Treatment with both extracts caused a reduction of these levels back to normal. Reduced glutathione (GSH) and Catalase activity (CAT) levels were significantly lower than normal in the Cisplatin group. Treatment with Urchin extracts increased the levels of GSH significantly above normal levels. NO, MDA, CAT, and GSH levels were significantly different from those of the cisplatin treated group (#### p-value < 0.0001). (n = 6, *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; **** p-value < 0.0001)

Fig. 5

Histological evaluation. Photomicrographs of brain sections showing the cerebral cortex of control rats (a, b, c) stained with hematoxylin and eosin (a) illustrating neuronal populations within the cortex are normally heterogeneous mixtures of many granular cells. Immuno-stained sections showed normal positive expression of Bcl2 in almost all apparent neurons (b), remarkably without apoptotic cells. Normal levels of expression of GFAP (c) immunostaining. Cisplatin-treated rats (d, e, f) showed neuronal degenerative changes particularly karyopyknosis and shrunken necrotic ghost like neurons. Bcl2 (e) immuno-stained sections showed negative Bcl2 indicating apoptotic cells. A strong positive GFAP (f) reaction and hypertrophic astrocytes indicating severe astrogliosis. Shell extract treated groups (100 mg/kg, g, h, i, and 50 mg/kg, j, k, l) showed intact homogenous neuropil with normal granular neurons and minimal reactive neuroglial cells infiltrates. Immuno-stained sections showed few apoptotic cells (high Bcl2 positive reaction), and mild astrogliosis. Spine extracts group 100 mg/kg (m, n, o) showed mild vacuolization of neuropil with pyknotic satellite cells and loss of normal pyramidal or fusiform shape of the neuron. Immuno-stained sections showed low positive reaction in most apparent neurons, more apoptotic cells and reactive astrogliosis evidenced. The 50 mg/kg spine treated group (p, q, r) showed normal pyramidal cells. Immuno-stained sections showed few apoptotic (Bcl2 positive) cells with normally distributed GFAP stained astrocytes

Fig. 6

Counting of the cerebral cortex neurons showed that Control rats showed an average of 8091 cells / mm³ compared to the Cisplatin group, which showed 3708 cells/ mm³. Sea Urchins Shell extract treatment showed an average of 5282 and 6293 cells / mm³ in the 50 mg and 100 mg groups respectively. Spine treated groups were showed higher neuron counts at 5057 and 6068 cells / mm³ in the 50 mg and 100 mg groups, respectively (n = 6, p-value < 0.0001). a Significantly different from the control group; p-value < 0.0001. b Significantly different from the Cisplatin model group; p-value < 0.0001. Data is expressed as mean ± SD, and Standard error of the mean SEM

Fig. 7

Immunohistochemical quantitative analysis showed the area of immuno-histochemical expression of GFAP (a) increased significantly after cisplatin administrations while the expression level of Bcl2 (b) decreased. Treatment with Spine (50–100) and Shell (50–100) extracts reversed these changes. Data presented as mean ± SD of the area (**** p < 0.0001). GFAP, and Bcl2 levels were significantly different from those of the cisplatin treated group (#### p-value < 0.0001)