Early maternal separation has mild effects on cardiac autonomic balance and heart structure in adult male rats

Figures & data

Figure 1.  Schematic diagram of the experimental protocol applied to control and maternally separated rats in adulthood. Transmitter implantation = surgical chronic implantation of transmitters for radiotelemetric ECG recordings. Pharmacological double blockade = pharmacological blockade of the two branches of the ANS with methylscopolamine (muscarinic receptor antagonist) and atenolol (beta-adrenergic receptor antagonist). IRS = daily exposure to restraint stress for 5 consecutive days. Rhythm recording = around-the-clock, radiotelemetric ECG recording for heart rate rhythmicity evaluation. Euthanized, heart and adrenals = euthanasia of the rats and subsequent removal of hearts and adrenals.

Table I.  Values (mean ± SEM) of average R–R interval (RR), time-domain HRV parameters (r-MSSD, pNN20), frequency-domain HRV parameters (LF, HF, LF/HF) during baseline conditions (30-min average value), restraint test (15 min), and recovery phase (45 min), in maternally separated (MS; n = 12) and control (n = 12) rats, at restraint tests 1 and 5.

Recording period		RR (ms)		r-MSSD (ms)		pNN20 (%)		LF (n.u.)		HF (n.u.)		LF/HF
	Group	Restraint 1	Restraint 5	Restraint 1	Restraint 5	Restraint 1	Restraint 5	Restraint 1	Restraint 5	Restraint 1	Restraint 5	Restraint 1	Restraint 5
Baseline (min)	MS	210.8 ± 4.43	211.8 ± 5.14	6.87 ± 0.45	7.58 ± 0.83	1.10 ± 0.22	1.87 ± 0.57	30.63 ± 2.53	29.88 ± 3.06	69.37 ± 2.53	70.12 ± 3.06	0.48 ± 0.07	0.42 ± 0.05
	Control	209.9 ± 3.45	207.7 ± 5.90	5.84 ± 0.68	5.50 ± 0.77	1.19 ± 0.32	1.06 ± 0.54	32.88 ± 3.38	32.04 ± 3.35	67.12 ± 3.38	67.96 ± 3.35	0.57 ± 0.10	0.56 ± 0.09
Test
1 (0–5)	MS	122.8 ± 0.97	125.5 ± 1.60	3.47 ± 0.77	2.24 ± 0.34	0.29 ± 0.16	0.22 ± 0.09	47.85 ± 2.85	47.89 ± 2.23	52.15 ± 2.85	52.11 ± 2.23	0.98 ± 0.11	0.96 ± 0.09
	Control	121.2 ± 1.08	122.1 ± 1.23	1.69 ± 0.17	2.07 ± 0.22	0.12 ± 0.08	0.10 ± 0.05	43.01 ± 1.84	48.06 ± 1.61	56.99 ± 1.84	51.94 ± 1.61	0.77 ± 0.06	0.90 ± 0.07
2 (5–10)	MS	122.3 ± 1.24	129.2 ± 1.92	1.88 ± 0.16	3.19 ± 0.30	0.06 ± 0.03	0.14 ± 0.04	54.01 ± 1.79	49.31 ± 2.36	45.99 ± 1.79	50.69 ± 2.36	1.15 ± 0.11	1.02 ± 0.10
	Control	123.2 ± 2.15	131.3 ± 2.97	2.12 ± 0.24	2.63 ± 0.29	0.20 ± 0.18	0.12 ± 0.06	51.02 ± 1.42	49.91 ± 2.01	48.98 ± 1.42	50.09 ± 2.01	1.01 ± 0.08	0.98 ± 0.10
3 (10–15)	MS	132.1 ± 1.98	139.1 ± 2.80	3.47 ± 0.31	4.09 ± 0.27	0.20 ± 0.07	0.29 ± 0.09	47.52 ± 2.46	46.76 ± 2.83	52.48 ± 2.46	53.24 ± 2.83	0.95 ± 0.09	0.93 ± 0.10
	Control	138.0 ± 2.24	145.4 ± 3.95	3.15 ± 0.37	3.45 ± 0.52	0.22 ± 0.07	0.36 ± 0.20	50.17 ± 2.33	47.29 ± 2.01	49.83 ± 2.33	52.71 ± 2.01	1.06 ± 0.11	0.80 ± 0.09
Recovery
4 (15–20)	MS	143.6 ± 2.80	147.0 ± 2.14	5.09 ± 0.47	4.64 ± 0.26	1.12 ± 0.32	1.16 ± 0.30	48.73 ± 3.69	47.18 ± 2.12	51.27 ± 3.69	52.82 ± 2.12	1.09 ± 0.19	0.93 ± 0.09
	Control	142.6 ± 2.60	145.4 ± 2.16	3.66 ± 0.42	3.75 ± 0.42	0.55 ± 0.20	0.50 ± 0.16	52.67 ± 1.67	54.88 ± 1.79	47.33 ± 1.67	45.12 ± 1.79	1.14 ± 0.07	1.15 ± 0.13
5 (20–25)	MS	152.1 ± 3.33	153.6 ± 2.70	4.79 ± 0.45	4.03 ± 0.28	0.84 ± 0.22	0.45 ± 0.08	52.62 ± 3.04	51.95 ± 2.50	47.38 ± 3.04	48.05 ± 2.50	1.08 ± 0.11	1.14 ± 0.10
	Control	146.8 ± 2.64	152.4 ± 1.98	3.68 ± 0.38	3.93 ± 0.54	0.53 ± 0.17	0.60 ± 0.21	53.35 ± 2.31	54.67 ± 2.31	46.65 ± 2.31	45.33 ± 2.31	1.19 ± 0.11	1.16 ± 0.15
6 (25–30)	MS	152.6 ± 3.41	154.5 ± 2.51	5.00 ± 0.43	4.32 ± 0.39	0.99 ± 0.26	0.60 ± 0.17	46.96 ± 3.62	45.34 ± 2.45	53.04 ± 3.62	54.66 ± 2.45	0.99 ± 0.16	0.87 ± 0.09
	Control	147.1 ± 2.92	154.9 ± 1.52	3.51 ± 0.24	4.03 ± 0.62	0.37 ± 0.10	0.57 ± 0.25	53.44 ± 2.20	50.73 ± 1.14	46.56 ± 2.20	49.27 ± 1.14	1.09 ± 0.15	1.00 ± 0.06
7 (30–35)	MS	154.5 ± 3.79	151.8 ± 2.20	4.46 ± 0.46	4.39 ± 0.36	0.98 ± 0.26	0.88 ± 0.22	44.34 ± 3.32	43.75 ± 3.24	55.66 ± 3.32	56.25 ± 3.24	0.77 ± 0.08	0.85 ± 0.13
	Control	151.7 ± 1.96	154.7 ± 3.03	4.07 ± 0.40	3.49 ± 0.35	1.10 ± 0.32	0.50 ± 0.18	50.15 ± 1.98	46.60 ± 2.35	49.85 ± 1.98	53.40 ± 2.35	1.04 ± 0.08	0.91 ± 0.08
8 (35–40)	MS	157.5 ± 3.85	160.5 ± 3.77	4.05 ± 0.39	4.53 ± 0.24	0.48 ± 0.15	0.42 ± 0.11	47.45 ± 2.85	46.72 ± 1.77	52.55 ± 2.85	53.28 ± 1.77	0.97 ± 0.12	0.91 ± 0.06
	Control	149.7 ± 2.30	159.8 ± 3.66	3.64 ± 0.43	4.19 ± 0.51	0.54 ± 0.25	0.55 ± 0.22	50.57 ± 2.71	44.33 ± 2.02	49.43 ± 2.71	57.67 ± 2.02	1.09 ± 0.13	0.82 ± 0.06
9 (40–45)	MS	158.8 ± 4.75	168.4 ± 5.72	4.06 ± 0.36	4.09 ± 0.28	0.34 ± 0.14	0.33 ± 0.11	42.18 ± 3.83	41.92 ± 3.64	57.82 ± 3.83	58.08 ± 3.64	0.82 ± 0.14	0.71 ± 0.09
	Control	158.4 ± 3.63	174.1 ± 5.65	4.49 ± 0.55	4.14 ± 0.51	0.72 ± 0.38	0.54 ± 0.15	43.14 ± 3.19	41.13 ± 3.35	56.86 ± 3.19	58.87 ± 3.35	0.73 ± 0.09	0.76 ± 0.09

Abbreviations: HF, high frequency; HRV, heart rate variability; LF, low frequency; r-MSSD, root mean square of successive differences between adjacent R–R intervals; n.u., normalized units; pNN20, percentage of successive interval differences larger than 20 ms.

Table II.  Values (mean ± SEM) of average R–R interval (RR), time-domain (r-MSSD, pNN20) and frequency-domain HRV parameters (LF, HF, LF/HF) in baseline conditions (30-min average value) and after scopolamine (15 min) and atenolol (45 min) injections, in maternally separated (MS; n = 12) and control (n = 12) rats, at pharmacological challenges 1 and 2 (tests 1 and 2).

Recording period		RR (ms)		r-MSSD (ms)		pNN20 (%)		LF (n.u.)		HF (n.u.)		LF/HF
	Group	Test 1	Test 2	Test 1	Test 2	Test 1	Test 2	Test 1	Test 2	Test 1	Test 2	Test 1	Test 2
Baseline (min)	MS	203.6 ± 3.89	205.9 ± 5.41	6.47 ± 0.70	6.01 ± 0.61	1.39 ± 0.38	1.72 ± 0.57	36.29 ± 3.50	31.55 ± 2.35	63.71 ± 3.50	68.45 ± 2.35	0.57 ± 0.07	0.50 ± 0.06
	Control	205.3 ± 4.23	200.7 ± 5.76	5.38 ± 0.56	5.60 ± 0.57	0.93 ± 0.26	1.57 ± 0.44	36.86 ± 1.96	38.78 ± 1.83	63.14 ± 1.96	61.22 ± 1.83	0.61 ± 0.05	0.64 ± 0.07
Scopolamine
1 (0–5)	MS	136.8 ± 2.51	138.5 ± 1.09	1.47 ± 0.17	1.69 ± 0.16	0.09 ± 0.04	0.05 ± 0.02	47.28 ± 2.39	48.34 ± 2.62	52.72 ± 2.39	51.66 ± 2.62	0.94 ± 0.09	0.99 ± 0.11
	Control	136.7 ± 3.65	137.1 ± 2.39	1.66 ± 0.20	1.73 ± 0.19	0.06 ± 0.02	0.08 ± 0.04	50.28 ± 1.95	48.36 ± 3.26	49.72 ± 1.95	51.64 ± 3.26	1.05 ± 0.08	1.01 ± 0.11
2 (5–10)	MS	133.5 ± 2.14	134.6 ± 2.05	0.78 ± 0.06	0.87 ± 0.07	0.00 ± 0.00	0.00 ± 0.00	48.48 ± 2.02	46.36 ± 2.21	51.52 ± 2.02	53.64 ± 2.21	0.98 ± 0.08	0.89 ± 0.08
	Control	135.8 ± 3.26	131.6 ± 2.22	0.90 ± 0.08	1.10 ± 0.13	0.01 ± 0.08	0.04 ± 0.02	49.05 ± 1.86	54.45 ± 2.94	50.95 ± 1.86	45.55 ± 2.94	0.94 ± 0.08	1.29 ± 0.13
3 (10–15)	MS	131.9 ± 1.70	135.8 ± 2.52	0.99 ± 0.14	0.76 ± 0.06	0.01 ± 0.01	0.00 ± 0.00	48.69 ± 3.29	48.49 ± 3.73	51.31 ± 3.29	51.51 ± 3.73	1.03 ± 0.12	0.94 ± 0.12
	Control	137.4 ± 3.15	133.5 ± 2.75	0.96 ± 0.16	0.95 ± 0.12	0.05 ± 0.04	0.00 ± 0.00	53.95 ± 2.32	58.59 ± 1.67	46.05 ± 2.32	41.41 ± 1.67	1.16 ± 0.13	1.37 ± 0.11
Atenolol
4 (15–20)	MS	155.2 ± 1.42	154.7 ± 1.28	1.79 ± 0.24	2.76 ± 0.45	0.28 ± 0.09	0.50 ± 0.19	54.32 ± 2.45	55.67 ± 2.34	45.68 ± 2.45	44.33 ± 2.34	1.18 ± 0.12	1.32 ± 0.12
	Control	155.2 ± 2.22	155.6 ± 1.77	1.93 ± 0.34	1.72 ± 0.19	0.26 ± 0.12	0.12 ± 0.03	48.24 ± 2.90	50.23 ± 3.27	51.76 ± 2.90	49.77 ± 3.27	1.00 ± 0.11	1.01 ± 0.12
5 (20–25)	MS	174.8 ± 2.69	173.6 ± 1.12	1.22 ± 0.14	1.57 ± 0.15	0.03 ± 0.02	0.02 ± 0.01	46.61 ± 1.56	42.17 ± 3.42	53.39 ± 1.56	57.83 ± 3.42	0.84 ± 0.07	0.72 ± 0.08
	Control	177.2 ± 2.26	174.0 ± 2.65	1.10 ± 0.07	1.15 ± 0.09	0.01 ± 0.01	0.00 ± 0.00	41.01 ± 2.30	42.91 ± 4.25	58.99 ± 2.30	57.09 ± 4.25	0.72 ± 0.06	0.84 ± 0.12
6 (25–30)	MS	185.5 ± 2.09	181.4 ± 1.87	1.25 ± 0.14	1.48 ± 0.15	0.04 ± 0.02	0.04 ± 0.04	46.43 ± 2.77	38.13 ± 3.16	53.57 ± 2.77	61.87 ± 3.16	0.91 ± 0.09	0.66 ± 0.08
	Control	185.2 ± 2.55	180.1 ± 2.86	1.38 ± 0.09	1.50 ± 0.15	0.02 ± 0.01	0.01 ± 0.01	38.28 ± 1.94	39.88 ± 4.84	61.72 ± 1.94	60.12 ± 4.84	0.64 ± 0.05	0.68 ± 0.11
7 (30–35)	MS	189.6 ± 2.62	185.5 ± 2.31	1.82 ± 0.30	1.60 ± 0.16	0.10 ± 0.05	0.08 ± 0.04	36.43 ± 3.86	37.53 ± 3.76	63.57 ± 3.86	62.47 ± 3.76	0.56 ± 0.07	0.66 ± 0.10
	Control	185.8 ± 1.68	182.1 ± 3.24	2.19 ± 0.21	1.66 ± 0.20	0.14 ± 0.05	0.07 ± 0.06	44.25 ± 3.22	42.95 ± 3.54	55.75 ± 3.22	57.05 ± 3.54	0.86 ± 0.11	0.76 ± 0.12
8 (35–40)	MS	191.0 ± 2.73	186.9 ± 2.42	1.65 ± 0.28	1.52 ± 0.13	0.07 ± 0.04	0.00 ± 0.00	41.67 ± 4.27	38.89 ± 4.01	58.33 ± 4.27	61.11 ± 4.01	0.83 ± 0.15	0.63 ± 0.08
	Control	187.5 ± 1.49	185.9 ± 3.22	1.50 ± 0.17	1.54 ± 0.16	0.04 ± 0.02	0.02 ± 0.01	33.21 ± 3.80	36.98 ± 2.52	66.79 ± 3.80	63.02 ± 2.52	0.49 ± 0.07	0.61 ± 0.05
9 (40–45)	MS	193.3 ± 2.63	190.1 ± 2.75	1.88 ± 0.24	1.42 ± 0.20	0.05 ± 0.02	0.01 ± 0.01	38.30 ± 4.36	33.74 ± 3.42	61.70 ± 4.36	66.26 ± 3.42	0.69 ± 0.10	0.55 ± 0.08
	Control	189.6 ± 2.20	188.2 ± 3.05	1.37 ± 0.11	1.34 ± 0.16	0.01 ± 0.01	0.00 ± 0.00	38.46 ± 2.59	35.26 ± 2.74	61.54 ± 2.59	64.74 ± 2.74	0.61 ± 0.07	0.58 ± 0.07
10 (45–50)	MS	196.5 ± 3.20	191.8 ± 2.39	1.68 ± 0.17	1.32 ± 0.14	0.09 ± 0.06	0.03 ± 0.03	45.18 ± 1.56	32.19 ± 5.57	54.82 ± 1.56	67.81 ± 5.57	0.78 ± 0.08	0.48 ± 0.11
	Control	191.7 ± 3.13	189.9 ± 3.22	1.44 ± 0.12	1.31 ± 0.11	0.08 ± 0.04	0.03 ± 0.02	41.89 ± 2.75	37.24 ± 5.12	58.11 ± 2.75	62.76 ± 5.12	0.64 ± 0.08	0.73 ± 0.17
11 (50–55)	MS	194.9 ± 2.93	193.6 ± 2.27	2.38 ± 0.23	1.29 ± 0.11	0.12 ± 0.04	0.01 ± 0.01	42.70 ± 5.19	31.07 ± 4.93	57.30 ± 5.19	68.93 ± 4.93	0.86 ± 0.13	0.55 ± 0.13
	Control	193.5 ± 3.53	189.5 ± 3.58	1.46 ± 0.14	1.37 ± 0.17	0.04 ± 0.02	0.00 ± 0.00	38.57 ± 3.61	42.77 ± 5.54	61.43 ± 3.61	57.23 ± 5.54	0.60 ± 0.08	0.79 ± 0.15
12 (55–60)	MS	196.7 ± 2.65	195.4 ± 2.76	2.10 ± 0.28	1.37 ± 0.10	0.11 ± 0.05	0.00 ± 0.00	34.98 ± 4.35	34.00 ± 5.00	65.02 ± 4.35	66.00 ± 5.00	0.70 ± 0.10	0.60 ± 0.12
	Control	196.9 ± 3.04	189.2 ± 3.77	1.27 ± 0.16	1.09 ± 0.14	0.00 ± 0.00	0.00 ± 0.00	29.37 ± 3.42	39.00 ± 5.22	70.63 ± 3.42	61.00 ± 5.22	0.46 ± 0.08	0.82 ± 0.19

Abbreviations: HF, high frequency; HRV, heart rate variability; LF, low frequency; r-MSSD, root mean square of successive differences between adjacent R–R intervals; n.u., normalized units; pNN20, percentage of successive interval differences larger than 20 ms.

Figure 2.  Daily rhythmicity of heart rate before and after the intermittent restraint protocol, in maternally separated and control rats. Average RR values (panel A) and r-MSSD values (panel C) for the 12 h light (open symbols) and dark phases (solid symbols) before (baseline: l and d) and after (post-stress: l1-l7 and d1-d7) the IRS. Delta values between each post-IRS day and the average pre-IRS value of light and dark phases are also reported (panels B and D), for both control (n = 12) and maternally separated (MS, n = 12) rats. RR, average R–R interval duration (ms). r-MSSD, root mean square of successive differences between adjacent R–R intervals (ms). Data are mean ± SEM. *p < 0.05, significant differences between MS and control animals (Student's t-test).

Table III.  Values (mean ± SEM) of gross cardiac characteristics, myocardial fibrosis in the LV and vascular distribution in the LV, in maternally separated (MS; n = 12) and control (n = 12) rats.

	Control	MS
LVW (mg)	889.5 ± 22.08	818.8 ± 20.17*
RVW (mg)	201.9 ± 7.01	201.8 ± 8.11
LVW/HW (mg/mg)	0.908 ± 0.005	0.802 ± 0.006*
RVW/HW (mg/mg)	0.192 ± 0.005	0.198 ± 0.006
LV chamber length (mm)	14.58 ± 0.27	14.20 ± 0.23
LV chamber equatorial  diameter (mm)	5.32 ± 0.18	5.02 ± 0.15
LV wall thickness (mm)	2.11 ± 0.06	2.14 ± 0.06
LV chamber volume (mm^3)	220 ± 15.67	189 ± 11.90*
Perivascular fibrosis (%)	0.316 ± 0.047	0.225 ± 0.043*
Interstitial fibrosis (%)	0.158 ± 0.060	0.243 ± 0.056*
Capillary density (n/mm^2)	132.2 ± 16.61	178.8 ± 11.42*
Venule density (n/mm^2)	6.31 ± 1.02	9.13 ± 2.54

Notes: LV, left ventricle; LVW, left ventricular weight; RVW, right ventricular weight; HW, heart weight; *p < 0.05: significant differences between MS and control rats (Student's t-test).