Dimethyl Fumarate as the Peripheral Blood Inflammatory Mediators Inhibitor in Prevention of Streptozotocin-Induced Neuroinflammation in Aged Rats

Figures & data

Figure 1 Representative flow cytometry graphs showing the cell surface marker analysis of peripheral blood and spleen lymphocyte populations (T, B, NK) and subsets of T lymphocytes (TCD3⁺CD4⁺CD8^−, TCD3⁺CD4⁻CD8⁺).

Notes: A and G graphs: FS vs SS plot of the whole blood (on the top) or spleen (on the bottom) with lymphocytes gated for analysis of T (CD3⁺), B (CD45RA⁺) and NK (CD161a⁺) lymphocytes (A graph), as well as TCD3⁺CD4⁺CD8⁻ and TCD3⁺CD4⁻CD8⁺ (G graph) lymphocyte subsets, circle highlights the lymphocyte population; B–D and H–J graphs show identification of different lymphocyte populations and T lymphocyte subsets based on the surface marker expression on lymphocytes. Histograms present the cell count (Y-axis) and fluorescent intensity (X-axis), unstained as well as TCD3⁺CD4⁺CD8⁻ and TCD3⁺CD4⁻CD8⁺ (G graph) lymphocyte subsets, circle highlights the lymphocyte population; B–D and H–J graphs show control cells (left) and cells stained (right) with antibody against the surface protein on lymphocytes: CD3-FITC (B and H graphs), CD161a-APC (C graph), CD45RA-PC7 (D graph), CD8-APC (I graph) and CD4-PC7 (J graph); E-F and K-L graphs show identification of different lymphocyte populations and T lymphocyte subsets based on the surface marker expression on lymphocytes. Cytograms present quadrant regions with analyzed lymphocyte populations and T lymphocyte subsets: NK cells (CD3⁻ CD161a⁺) (E graph), B lymphocytes (CD3⁻ CD45RA⁺) and T lymphocytes (CD3⁺ CD45RA⁻) (F graph) as well as T CD4⁺ lymphocytes (CD3⁺ CD4⁺CD8⁻) (K graph) and T CD8⁺ lymphocytes (CD3⁺CD4⁻CD8⁺) (L graph).

Abbreviations: FITC, fluorescein isothiocyanate; PC7, phycoerythrin cyanin 7; APC, allophycocyanin.

Table 1 The Total Number of White Blood Cells (WBC), Percentage and Total Numbers of Lymphocytes, Neutrophils, Eosinophils, Basophils and Monocytes, Relative Thymus and Spleen Weight in Young Rats Subjected to Dimethyl Fumarate (DMF) or Control Therapy (CTR) Initiated on Day 0 (0.4% DMF or Standard Rat Chow) and Intracerebroventricular Injection of Streptozotocin (STZ) or Vehicle (VEH) on Days 2 and 4

Group Parameter	VEH CTR Mean ± SD	VEH DMF Mean ± SD	STZ CTR Mean ± SD	STZ DMF Mean ± SD
WBC	4098.42 ± 1080.03	3780.56 ± 1203.24	3622.35 ± 1432.47	4471.54 ± 953.52 ^&; #
Lymphocytes
%	76.59 ± 6.60	81.24 ± 4.09 ^$	75.41 ± 7.19	77.41 ± 9.33
No./µL	3138.98 ± 996.71	3071.33 ± 981.41	2731.61 ± 1295.41	3461.42 ± 1560.67
Neutrophils
%	16.13 ± 5.36	14.35 ± 4.72	17.25 ± 4.88	14.86 ± 5.08 ^#
No./µL	661.08 ± 213.64	542.51 ± 219.30	624.86 ± 291.78	664.47 ± 209.44 ^&
Eosinophils
%	1.50 ± 0.63	1.50 ± 0.65	1.58 ± 0.61	1.27 ± 0.47
No./µL	61.48 ± 21.38	56.71 ± 22.74	57.23 ± 21.51	56.79 ± 20.01
Basophils
%	3.14 ± 1.21	3.60 ± 1.17	4.11 ± 1.69	2.55 ± 1.13 ^$;&
No./µL	128.69 ± 68.43	136.10 ± 48.52	148.88 ± 34.42 ^$$	114.02 ± 39.11
Monocytes
%	3.33 ± 1.40	2.31 ± 1.01 ^$	3.07 ± 1.14	2.08 ± 1.04 ^#
No./µL	136.48 ± 53.20	87.33 ± 20.77 ^$	111.21 ± 53.96	93.01 ± 35.16 ^&; #
Relative thymus weight (mg/100g b.w.)	93.61 ± 19.04	93.37 ± 15.37	95.16 ± 23.79	102.19 ± 27.26
Relative spleen weight (mg/100g b.w.)	200.02 ± 29.58	206.06 ± 34.64	205.53 ± 31.80	196.02 ± 36.96

Notes: Data is presented as a mean ± SD and was analyzed using Kruskal–Wallis or Mann–Whitney-U test; ^$P < 0.05 and ^$$P < 0.01 indicate significance of differences vs VEHCTR; ^&P < 0.05 represents significance of differences vs VEHDMF; ^#P < 0.05 indicates significance of differences vs STZCTR.

Table 2 The Total Number of White Blood Cells (WBC), Percentage and Total Numbers of Lymphocytes, Neutrophils, Eosinophils, Basophils and Monocytes, Relative Thymus and Spleen Weight in Aged Rats Subjected to Dimethyl Fumarate (DMF) or Control Therapy (CTR) Initiated on Day 0 (0.4% DMF or Standard Rat Chow) and Intracerebroventricular Injection of Streptozotocin (STZ) or Vehicle (VEH) on Days 2 and 4

Group Parameter	VEH CTR Mean ± SD	VEH DMF Mean ± SD	STZ CTR Mean ± SD	STZ DMF Mean ± SD
WBC	7218.18 ± 1479.28	7954.44 ± 1322.98 ***	6524.61 ± 1514.33 ***	5465 ± 2169.26 ^&
Lymphocytes
%	63.92 ± 6.24	53.43 ± 12.28 ***	64.28 ± 12.74 *	43.34 ± 9.67 ^#;***
No./µL	4613.86 ± 1068.32	4250.1 ± 579.26 **	4194.02 ± 731.59	2368.53 ± 839.68 ^#;***
Neutrophils
%	30.67 ± 6.18 ***	40.39 ± 7.07 ^$; ***	41.88 ± 6.14 ^$;***	51.15 ± 12.12 ^&; ***
No./µL	2213.82 ± 420.89 ***	3212.8 ± 910.49 ***	2732.51 ± 896.41 ^$$; ***	2795.35 ± 814.98 ***
Eosinophils
%	1.19 ± 0.37	1.60 ± 0.65	0.88 ± 0.20 **	1.34 ± 0.46
No./µL	85.9 ± 17.20	127.27 ± 30.99 ***	57.42 ± 8.48	73.23 ± 20.75 *
Basophils
%	22.15 ± 2.2 **	21.5 ± 3.5 **	20.30 ± 0.20 *	12.41 ± 4.34 ***^; #; &
No./µL	1598.83 ± 158.8	1710.21 ± 86.55 **	1324.5 ± 255.03 *	678.21 ± 39.15 **
Monocytes
%	1.43 ± 0.47 **	3.56 ± 0.89 *	1.62 ± 0.46 ***	7.28 ± 2.99 ***^; &&; #
No./µL	103.22 ± 40.38	283.18 ± 64.97 ***	105.7 ± 21.98	397.85 ± 25.06^#
Relative thymus weight (mg/100g b.w.)	38.30 ± 12.05 ***	25.66 ± 8.92 ***^; $	31.14 ± 6.90 ***	36.31 ± 9.79 ***^; &
Relative spleen weight (mg/100g b.w.)	203.66 ± 43.65	194.69 ± 43.70 ^$	205.60 ± 33.13	217.26 ± 48.63 ^&

Notes: Data is presented as a mean ± SD and was analyzed using Kruskal–Wallis or Mann–Whitney-U test; ^$P < 0.05 and ^$$P < 0.01 indicates significance of differences vs VEHCTR; ^&P < 0.05 and ^&&P < 0.01 indicate significance of differences vs VEHDMF; ^#P < 0.05 represents significance of differences vs STZCTR; *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significance of differences between the corresponding group of young and aged rats.

Figure 2 The percentage and total number of T (CD3⁺), B (CD3⁻CD45RA⁺), NK (CD3⁻CD161a⁺) lymphocytes in the peripheral blood mononuclear cells (PBMC) analyzed by flow cytometric method in aged rats subjected to dimethyl fumarate (DMF) or control therapy (CTR) initiated on day 0 (0.4% DMF or standard rat chow) and intracerebroventricular injection of streptozotocin (STZ) or vehicle (VEH) on days 2 and 4.

Notes: Data is presented as mean ± SD and was analyzed using Kruskal–Wallis or Mann–Whitney-U test; ^#P < 0.05 and ^##P < 0.01 indicate significance of differences vs VEHDMF; ^$$P < 0.01 represents significance of differences vs STZCTR; ^&&P < 0.01 indicates significance of differences vs VEHCTR.

Figure 3 The percentage and total number of TCD4⁺ (CD3⁺CD4⁺CD8⁻) and TCD8⁺ (CD3⁺CD4⁻CD8⁺) lymphocytes in the peripheral blood mononuclear cells (PBMC) analyzed by flow cytometric method in aged rats subjected to dimethyl fumarate (DMF) or control therapy (CTR) initiated on day 0 (0.4% DMF or standard rat chow) and intracerebroventricular injection of streptozotocin (STZ) or vehicle (VEH) on days 2 and 4.

Notes: Data is presented as mean ± SD and was analyzed using Kruskal–Wallis or Mann–Whitney-U test; ^#P < 0.05 and ^##P < 0.01 indicate significance of differences vs VEHDMF; ^$P < 0.05 and ^$$P < 0.01 represent significance of differences vs STZCTR; ^&P < 0.05 and ^&&P < 0.01 indicate significance of differences vs VEHCTR.

Table 3 The Complete Blood Count: Red Blood Cells (RBC), Platelets (PLT), Haemoglobin Concentration (HGB), Mean Haemoglobin Concentration in the Red Blood Cell (MCHC), Mean Mass of the Haemoglobin in the Red Blood Cell (MCH), Mean Corpuscular Volume (MCV), Hematocrit (HCT), Mean Platelet Volume (MPV), Red Cell Distribution Width (RDW) in Young (y) and Aged (a) Rats Subjected to Dimethyl Fumarate (DMF) or Control Therapy (CTR) Initiated on Day 0 (0.4% DMF or Standard Rat Chow) and Intracerebroventricular Injection of Streptozotocin (STZ) or Vehicle (VEH) on Days 2 and 4

Group Parameter	VEH DMFy Mean ± SD	VEH DMFa Mean ± SD	STZ CTRy Mean ± SD	STZ CTRa Mean ± SD	STZ DMFy Mean ± SD	STZ DMFa Mean ± SD
RBC (No.)x10^3	8268.89 ± 337.43	8567.42 ± 546.11	8217.00 ± 471.22	8480.00 ± 20.00	8145.56 ± 470.51	8054.17 ± 595.73 ^##; &
PLT (No.)x10^3	518.63 ± 65.90	598.25 ± 108.62 *	421.60 ± 91.67	661.50 ± 11.50 **	365.00 ± 131.54 ^&	630.45 ± 95.62 **
HGB (g/dL)	14.28 ± 0.38	14.90 ± 0.23 **	14.06 ± 0.75	15.35 ± 0.15 **	14.16 ± 0.62	13.90 ± 1.42 ^#
MCHC (g/dL)	31.91 ± 0.60	31.87 ± 0.56	31.86 ± 0.36	31.90 ± 0.40	31.83 ± 0.68	32.40 ± 0.63 *
MCH (pg)	17.20 ± 0.49	16.90 ± 0.62	17.14 ± 0.58	17.95 ± 0.05 *	17.38 ± 0.34	17.29 ± 0.81
MCV (fL)	54.43 ± 1.41	53.45 ± 1.71	53.75 ± 1.85	55.15 ± 0.55 *	54.61 ± 1.26	54.13 ± 3.02
HCT (%)	44.91 ± 1.42	46.75 ± 1.31 *	44.15 ± 2.66	48.35 ± 1.15 *	44.49 ± 2.78	43.07 ± 3.26 ^#; &
MPV (fL)	4.89 ± 0.66	4.58 ± 0.39	4.65 ±0.55	4.55 ± 0.05	5.11 ± 0.70	4.18 ± 0.31 **^; #
RDW (%)	14.08 ± 0.81	16.22 ± 1.30 *	14.98 ± 0.82	16.28 ± 0.25 **	14.08 ± 0.81 ^$	15.65 ± 0.80 ***

Notes: Data is presented as a mean ± SD and was analyzed using Kruskal–Wallis or Mann–Whitney-U test; ^$P < 0.05 indicates significance of differences vs VEHCTR of the same age; ^&P < 0.05 represents significance of differences vs VEHDMF of the same age; ^#P < 0.05 and ^##P < 0.01 indicate significance of differences vs STZCTR of the same age; *P < 0.05,**P < 0.01, and ***P < 0.001 represent significance of difference between the corresponding group of young and aged rats.

Figure 4 Plasma concentration of interleukin 6 (IL-6) (A) and interleukin 10 (IL-10) (B) in young and aged rats subjected to dimethyl fumarate (DMF) or control therapy (CTR) initiated on day 0 (0.4% DMF or standard rat chow) and intracerebroventricular injection of streptozotocin (STZ) or vehicle (VEH) on days 2 and 4.

Notes: Data is presented as mean ± SD and was analyzed using Kruskal–Wallis or Mann–Whitney-U test; *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significance of difference between the corresponding group of young and aged rats; ^#P < 0.05 represents significance of difference vs the VEHDMF group of the same age; ^$P < 0.05 indicates significance of differences vs the STZCTR group of the same age.

Figure 5 Concanavalin (Con) A-stimulated splenocyte mononuclear cell (SMC) production of interferon (IFN)-γ (A) and interleukin (IL)-10 (B) in young rats subjected to dimethyl fumarate (DMF) or control therapy (CTR) initiated on day 0 (0.4% DMF or standard rat chow) and intracerebroventricular injection of streptozotocin (STZ) or vehicle (VEH) on days 2 and 4.

Notes: Data is presented as mean ± SD and was analyzed using Kruskal–Wallis or Mann–Whitney-U test; ^#P < 0.05 indicates significance of differences vs non-stimulated (control) SMC; ^P < 0.05 represents significance of differences vs STZCTR after Con-A stimulation; *P < 0.05 indicates significance of differences vs VEHDMF after Con-A stimulation; ^$P < 0.05 represents significance of differences vs VEHCTR after Con-A stimulation.

Figure 6 Concanavalin (Con) A-stimulated splenocyte mononuclear cell (SMC) production of interleukin (IL)-6 (A) and interleukin (IL)-10 (B) in aged rats subjected to dimethyl fumarate (DMF) or control therapy (CTR) initiated on day 0 (0.4% DMF or standard rat chow) and intracerebroventricular injection of streptozotocin (STZ) or vehicle (VEH) on days 2 and 4.

Notes: Data is presented as mean ± SD and was analyzed using Kruskal–Wallis or Mann–Whitney-U test; ^#P < 0.05 indicates significance of differences vs non-stimulated (control) SMC; ^$P < 0.05 and ^$$P < 0.01 represent significance of differences vs VEHCTR after Con-A stimulation; ^@P < 0.05 indicates significance of differences vs VEHCTR control.

Figure 7 Concanavalin (Con) A-stimulated peripheral blood mononuclear cell (PBMC) production of interferon (IFN)-γ in young rats (A) and interleukin (IL)-10 in aged rats (B) subjected to dimethyl fumarate (DMF) or control therapy (CTR) initiated on day 0 (0.4% DMF or standard rat chow) and intracerebroventricular injection of streptozotocin (STZ) or vehicle (VEH) on days 2 and 4.

Notes: Data is presented as mean ± SD and was analyzed using Kruskal–Wallis or Mann–Whitney-U test; ^#P < 0.05 indicates significance of differences vs non-stimulated (control) PBMC; ^$P < 0.05 represents significance of differences vs VEHCTR after Con-A stimulation; ^@P < 0.05 indicates significance of differences vs VEHCTR control.

Figure 8 Plasma concentration of corticosterone in young and aged rats subjected to dimethyl fumarate (DMF) or control therapy (CTR) initiated on day 0 (0.4% DMF or standard rat chow) and intracerebroventricular injection of streptozotocin (STZ) or vehicle (VEH) on days 2 and 4.

Notes: Data is presented as mean ± SD and was analyzed using Kruskal–Wallis or Mann–Whitney-U test; **P < 0.01 indicates significance of difference between the corresponding group of young and aged rats; ^#P < 0.05 and ^##P < 0.01 represent significance of difference vs the VEHDMF group of the same age; ^&P < 0.05 and ^&&P < 0.01 represent significance of differences vs the VEHCTR group of the same age.