The possible role of cerium oxide (CeO₂) nanoparticles in prevention of neurobehavioral and neurochemical changes in 6-hydroxydopamine-induced parkinsonian disease

Peer review under responsibility of Alexandria University Faculty of Medicine.

Available online 20 January 2017

Figures & data

Fig. 2 Comparison between the different groups according to Dopamine HPLC (ng/mg tissue).

Fig. 3 Comparison between the different groups according to TAC (mM/mg protein).

Fig. 4 Comparison between the different groups according to MDA (nM/g tissue).

Fig. 5 Comparison between the different groups according to Nitrite (μM/mg protein).

Fig. 7 Correlations between dopamine levels, oxidative stress markers (TAC, MDA & Nitrite) and caspase 3 measured in striatal tissue of all studied groups.

Table 1 Neuro-behavior finding in control, 6-OHDA untreated and preventive groups 3 weeks after intrasrtiatal injection of 6-OHDA.

	Controls	6-OHDA	CeO2NPs –preventive group	KWχ^2	P
Open field parameters
Latency to move in (seconds)	4 (3–6.5)	84 (70–95)^$$	31.5(23–40.5)^$$^,^##	38.587	<0.001
Number of crossed squares	25 (19.3–29)	9 (6–10)^$$	17.5(12.8–20.5)^$^,^##	34.398	<0.001
Latency to rear (seconds)	10 (7.8–16.5)	19.5 (12–43)^$	11.5 (10–17.3)^#	8.898	0.012
Number of rears	13.5 (8–15)	5 (3–7.8)^$$	8 (5.8–8.8)^$^,^#	19.898	<0.001
Rotarod test				F	P
Mean time of latency to fall^a	93.7 ± 17.2	32.4 ± 5.94^$$	52.9 ± 5.92^$$^,^##	199.900	<0.001
Stepping test				KWχ^2	P
Total Steps taken by Rt forelimb^b	20.5 (18.8–23.5)	3.0(1.3–5)^$$	7.0(5.8–9.3)^$$^,^##	35.848	<0.001
Total Steps taken by Lt forelimb^b	21.5 (19.8–22.8)	22.0(19–24)^**	19.5 (15–22)^*	3.621	0.164
Time to initiate steps by Rt forelimb^a	4.0(3.4–5.1)	29.6(25.1–35)^$$	16.1(15.3–18.5)^$$^,^##	39.399	<0.001
Time to initiate steps by Lt forelimb^a	4.67 (3.2–6.2)	5.50 (4.3–6.6)^**	6.67 (5.2–7.7)^*	4.354	0.113

Data were expressed as medians (inter-quartile range) and analyzed by non–parametric ANOVA (Kruskal Wallis), followed when significant by Mann Whitney test, except for mean time of latency to fall in rotarod test, where data were expressed as Mean ± SD and analyzed by (one way ANOVA), followed by Post-Hoc Tukey test. Each group contained 10 rats.

a Data represented mean of three trials for each rat.

b Data were expressed as the sum of forehand and backhand steps made by each forepaw.

$ P ⩽ 0.05.

$$ P ⩽ 0.001versus control group.

# P ⩽ 0.05.

## P ⩽ 0.001 versus 6-OHDAgroup. Wilcoxon signed ranks test for comparing between right and left forelimb in each group.

* p ⩽ 0.05.

** p ⩽ 0.001 versus opposite limb of the same group.

Table 2 Neuro-behavior findings in control, 6-OHDA untreated group and preventive group 6 weeks after intrastriatal injection of 6-OHDA.

	Controls	6-OHDA untreated group	CeO2NPs –preventive group	KWχ^2	p
Latency to move in second	4.0(2–6)	70.50(45.8–84.3)^$$	31.0(22.3–43.5)^$$^,^#	23.906^*	<0.001^*
Squares crossed	24.0(19–28.8)	8.0(4–9.3)^$$	17.0(10.8–20.3)^$^,^#	18.951^*	<0.001^*
Latency to rear in second	12.50(7–19.5)	36.0(18.3–45.5)^$	21.50(19.3–26.8)^$	8.115^*	0.017^*
No of rear	13.0(7.8–15.5)	3.50(2–4.3)^$$	5.50(3.8–9)^$	17.722^*	<0.001^*
Rotarod test				F	P
Mean time of latency to fall^a	101.10 ± 21.44	30.27^$$ ± 8.02	50.07^$$^,^# ± 7.30	69.431^*	<0.001^*
Stepping test				KWχ^2	p
Mean Total Steps have taken by RT forelimb^b	22.0(19.8–34.5)	1.0(0.0–2)^$$	7.0(6.5–8.3)^$$^,^##	25.980^*	<0.001^*
Mean Total Steps have taken by LT forelimb^b	25.50(21.5–37)	28.50(11.8–30.5)	26.0(17–27.8)	0.713	0.700
^WRSTp	0.118	0.005^*	0.005^*

Data were expressed as medians (inter-quartile range) and analyzed by non–parametric ANOVA (Kruskal Wallis), followed when significant by Mann Whitney test, except for mean time of latency to fall in rotarod test, where data were expressed as Mean ± SD and analyzed by (one way ANOVA), followed by Post-Hoc Tukey test. Each group contained 10 rats.

^**p ⩽ 0.001 versus opposite limb of the same group.

a Data represented mean of three trials for each rat.

b Data were expressed as the sum of forehand and backhand steps made by each forepaw.

$ P ⩽ 0.05,

$$ P ⩽ 0.001 versus control group,

# P ⩽ 0.05,

## P ⩽ 0.001 versus 6-OHDAgroup. Wilcoxon signed ranks test for comparing between right and left forelimb in each group.

* p ⩽ 0.05,

Table 3 Neuro-behavior findings in the preventive group 3 and 6 weeks after intrastriatal injection of 6-OHDA.

	After 3 weeks (n = 10)	After 6 weeks (n = 10)	Test of Sig.	p
Latency to move (seconds)	31.50 (23.0–45.0)	31.0 (20.0–50.0)	Z = 0.877	0.380
Number of crossed Squares	17.50 (11.0–23.0)	17.0 (5.0–22.0)	Z = 0.842	0.400
Latency to rear in (seconds)	11.50 (4.0–32.0)	21.50 (17.0–33.0)	Z = 2.536^*	0.011^*
Number of rears	8.0 (4.0–11.0)	5.50 (0.0–10.0)	Z = 1.196	0.232
Mean time of latency to fall	52.97 ± 1.87	50.07 ± 2.31	t = 3.728^*	0.005^*
Total Steps taken by RT forelimb	7.0 (4.0–11.0)	7.0 (4.0–10.0)	Z = 0.272	0.785
Total Steps taken by LT forelimb	19.50 (14.0–24.0)	26.0 (20.0–44.0)	Z = 2.366^*	0.018^*
Time to initiate steps by RT forelimb	16.17 (15.0–20.0)	22.33 (15.67–30.0)	Z = 2.805^*	0.005^*
Time to initiate steps by LT forelimb	6.67 (3.90–8.33)	5.67 (4.33–7.67)	Z = 1.073	0.283

t: Paired t-test

Z: Z for Wilcoxon signed ranks test

* Statistically significant at p ⩽ 0.05

Fig. 1 Open field parameters: (A) latency to move (seconds), (B) number of crossed squares (C) latency to rear and (D) number of rears, three weeks following intrastriatal injection 6-OHDA injection. Data are expressed as medians (intra-quartile rang), ^$ P ⩽ 0.05, ^$$ P ⩽ 0.001versus control group, ^# P ⩽ 0.05, ^## P ⩽ 0.001 versus 6-OHDAgroup.

Fig. 6 Comparison between the different studied groups according to ratio of CASPASE 3 activity.

Table 4 Correlation between neurobehavioral findings and biochemical markers in all studied rats.

		Dopamine	TAC	MDA	Nitrite	Caspase 3
Latency to move (s)	r1	−0.741^**	−0.774^**	0.720^**	0.497^**	0.734^**
	P	<0.001^**	<0.001^**	<0.001^**	0.005^**	<0.001^**
Number of crossed Squares	r1	0.663^**	0.708^**	−0.724^**	−0.470^**	−0.613^**
	P	<0.001^**	<0.001^**	<0.001^**	0.009^**	<0.001^**
Latency to rear in (s)	r1	−0.289	−0.487^**	0.506^**	0.546^**	0.367^*
	P	0.121	0.006^**	0.004^**	0.002^**	0.046^*
Number of rears	r1	0.633^**	0.731^**	−0.544^**	−0.565^**	−0.654^**
	P	<0.001^**	<0.001^**	0.002^**	0.001^**	<0.001^**
Mean time of latency to fall	r2	0.827^**	0.822^**	−0.670^**	−0.456^*	−0.767^**
	P	<0.001^**	<0.001^**	<0.001^**	<0.001^**	<0.001^**
Total Steps taken by RT forelimb	r1	0.720^**	0.905^**	−0.723^**	−0.542^**	−0.819^**
	P	<0.001^**	<0.001^**	<0.001^**	0.002^**	<0.001^**
Total Steps taken by LT forelimb	r1	0.122	0.284	0.036	0.081	−0.035
	P	0.521	0.128	0.852	0.670	0.856
Time to initiate steps by RT forelimb	r1	−0.804^**	−0.840^**	0.810^**	0.533^*	0.682^**
	P	<0.001^**	<0.001^**	<0.001^**	<0.001^**	<0.001^**
Time to initiate steps by LT forelimb	r1	−0.138	−0.065	0.276	0.038	0.145
	P	0.467	0.733	0.141	0.840	0.446

r₁: Spearman coefficient correlation, r₂: Pearson coefficient,

* p ⩽ 0.05.

** p ⩽ 0.01. spearman coefficient correlation between neurobiochemical markers and neurobehavioral findings in all studied groups (n = 60) except the mean time of latency to fall with biochemical markers assessed by pearson correlation in all studied groups (n = 60).