Melatonin and the cardiovascular system in animals: systematic review and meta-analysis

Veiga, Eduardo Carvalho de Arruda; Simões, Ricardo dos Santos; Caviola, Leonardo L.; Abreu, Luiz Carlos; Cavalli, Ricardo Carvalho; Cipolla-Neto, José; Baracat, Edmund Chada; Soares Júnior, José Maria

doi:10.6061/clinics/2021/e2863

Eduardo Carvalho de Arruda Veiga ^**Corresponding author. E-mail: eduveiga56@gmail.com

Departamento de Obstetricia e Ginecologia, Hospital das Clinicas HCFMRP-USP, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, SP, BR.

http://orcid.org/0000-0002-0167-861X

Ricardo dos Santos Simões

Departamento de Obstetricia e Ginecologia, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR.

http://orcid.org/0000-0001-5916-0015

Leonardo L. Caviola

Departamento de Obstetricia e Ginecologia, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR.

http://orcid.org/0000-0001-9301-0416

Luiz Carlos Abreu

Disciplina de escrita cientifica, Faculdade de Medicina do ABC, Santo Andre, SP, BR.

http://orcid.org/0000-0002-7618-2109

Ricardo Carvalho Cavalli

Departamento de Obstetricia e Ginecologia, Hospital das Clinicas HCFMRP-USP, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, SP, BR.

http://orcid.org/0000-0001-5010-4914

José Cipolla-Neto

Departamento de Fisiologia e Biofisica, Instituto de Ciencias Biomedicas (ICB-USP), Universidade de Sao Paulo, Sao Paulo, SP, BR.

http://orcid.org/0000-0003-3748-3731

Edmund Chada Baracat

Departamento de Obstetricia e Ginecologia, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR.

http://orcid.org/0000-0003-0111-9030

José Maria Soares Júnior

Departamento de Obstetricia e Ginecologia, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR.

http://orcid.org/0000-0003-0774-9404

*Corresponding author. E-mail: eduveiga56@gmail.com

No potential conflict of interest was reported.

Veiga ECA contributed substantially to the study conception and design, definition of intellectual content, was involved in literature search, data analysis, statistical analysis, and manuscript preparation, drafting and critical review for important intellectual content, and approved the final manuscript version to be published. Simões RS, Caviola LL, Abreu LC and Cavalli RC were involved in data analysis and statistical analysis, manuscript drafting and critical review for important intellectual content, and approved the final manuscript version to be published. Cipolla-Neto J, Baracat EC and Soares Junior JM substantially contributed to the study conception and design, definition of intellectual content, were involved in manuscript preparation, drafting and critical review for important intellectual content, and approved the final manuscript version to be published.

Authors	Animal type	Animal race	Age (months)	Weight	Induction model	Site injury
Zhang et al. (21)	Mice	C57/B6	-	-	Sepsis-induced cardiac dysfunctional	Cardiovascular system
Benova et al. (22)	Rat	Wistar	9 months	-	Obesity	Cardiovascular system
Chen et al. (23)	Rat	Sprague-Dawley	-	200-250 g	Myocardial ischemia reperfusion	Myocardial tissue
Liu et al. (24)	Mice	C57/B6	6 months	-	Myocardial infarction	Heart
Simko et al. (25)	Rat	Wistar	3 months	-	hypertension	Cardiovascular system
Simko et al. (26)	Rat	Wistar	3 months		hypertension	Cardiovascular system
Stacchiotti et al. (27)	Mice	B6.V^LEAN/OlaHsd and B6.V-Lep^ob/OlaHsd	4 weeks	-	Obesity	Mitochondria of cardiomyocyte
Chaudagar et al. (28)	Rat	Wistar	8 months	-	hypertension	Cardiovascular system
Salmanoglu et al. (29)	Rat	Wistar	-	250-350 g	Diabetic	Liver tissue
Cheng et al. (30)	Rabbits	New Zealand	4 weeks	2.0-2.5 kg	Atherosclerosis	Aorta
Liu et al. (31)	Rat	Sprague-Dawley	3 months	280-360 g	Myocardial ischemia reperfusion	Myocardial tissue
Zhu et al. (32)	Rat	Sprague-Dawley	10 weeks	250 g	Myocardial infarction	Heart
Liu et al. (33)	Rat	-	-	350-400 g	Myocardial ischemia reperfusion	Heart
Drobnik et al. (34)	Rat	Wistar	4 weeks	290-320 g	Myocardial infarction	Heart
Repova et al. (35)	Rat	Wistar	3 months		hypertension	Cardiovascular system
Drobnik et al. (36)	Rat	Wistar	-	300-330 g	Myocardial infarction	Heart
Chen et al. (37)	Mice	Mice Gpx^-/- C57BL/6	-	-	Myocardial ischemia reperfusion in vitro	Heart
Petrosillo et al. (38)	Rat	Wistar	-	250-330 g	Myocardial ischemia reperfusion	Heart

Authors	Sample size	Number of groups	Number of animals/groups	Melatonin administration	Melatonin doses	Dependent variables
Zhang et al. (21)	24	4	6	Intraperitoneal injection	30 mg/kg	Echo, histological analysis, creatinine kinase measurement, TUNEL analysis, western blotting.
Benova et al. (22)	48	4	12	Drinking water	10 mg of melatonin was dissolved in 100 mL of water for 8 weeks	Heart function in Langendorff perfusion, western blot, real-time PCR,
Chen et al. (23)	30	5		Intraperitoneal at the reperfusion	20 mg/kg	Echo, IS2, lactate dehydrogenase release, CMEC measurement in vitro IRI assay, western blotting, qRT-PCR, and detection of autophagosomes.
Liu et al. (24)	18	3	6	Gavage	50 mg/kg	Echo, histological analysis, PCR, western blot, CTRP3 detection.
Simko et al. (25)	40	4	10	Water consumption was 12-13 mL/100 g of body weight	10 mg of melatonin was dissolved in 100 mL of water for 4 weeks	Hemodynamics measures, biometric analysis, determination of hydroxyproline, angiotensin, and aldosterone analysis.
Simko et al. (26)	66	6	11	Drinking water adjustment to daily water consumption to ensure the correct dosage	10 mg/kg/ day for 6 weeks	Hemodynamics measures, determination of hydroxyproline, NO synthase activity, oxidative load measurement, and western blotting of NF-_ΚB.
Stacchiotti et al. (27)	40	4	10	5^th to 13^th weeks of life/drinking water	100 mg/kg/day for 8 weeks	Histomorphometric evaluations, nuclear cardiomyocyte morphometry, mitochondrial and immunohistochemical analysis.
Chaudagar et al. (28)	24	4	6	Drinking water	10 mg/kg/day for 67 days	Hemodynamics measures, biometric analysis, and NO assays.
Salmanoglu et al. (29)	35	6	-	Oral gavage	10 mg/kg/day for 2 weeks	Vasocontractile response, measurement of total cholesterol, LDL, HDL, glucose, NO, and insulin, MDA assay, and tissue antioxidant levels.
Cheng et al. (30)	60	3	20	-	20 mg/kg for 4 weeks	Immunohistochemical analysis, HE staining, western blot analysis, and qRT-PCR.
Liu et al. (31)	60	5	12	Intravenous injection immediately after reperfusion	10 mg/kg	IF2, myocardial ultrastructure, western blotting and determination of the opening degree of MPTPs.
Zhu et al. (32)	-	-	-	Melatonin stem cells were treated for 24 hours	5 µM	Measurements of cell culture antioxidant properties, apoptosis, analysis of paracrine factors, LV functions, histology.
Liu et al. (33)	60	6	12	Intraperitoneal injection	Group I: 2.5 mg/kg, Group II: 5 mg/kg, Group III: 10 mg/kg	Hemodynamics measures, apoptosis, electron microscope examination, analysis on mitochondria.
Drobnik et al. (34)	21	3	7	Drinking water for 6 weeks	10 mg/kg	Collagen determination, estimation of glycosaminoglycans, electron microscope examination.
Repova et al. (35)	40	4	10	Drinking water for 6 weeks	10 mg/kg	Collagen determination, hemodynamics measures.
Drobnik et al. (36)	60	5	12	Intraperitoneal injection for 4 weeks	Group 1: 300 µg/100 g b.w. Group 4: 3 mg/100 g.b.w. Group 5: 1.5 mg/100 g.b.w.	Estimation of lipid peroxidation, collagen determination, estimation of glycosaminoglycans.
Chen et al. (37)	-	-	-	Intraperitoneal injection 30 min before harvesting the hear for in vitro preparation	150 µg/kg	Cardiac function, hemodynamics measures, lactate dehydrogenase released, apoptosis, immunohistochemistry.
Petrosillo et al. (38)	42	6	7	Krebs-Henseleit solution for isolated heart	50 µM	Infarct size, lactate dehydrogenase released, hemodynamics measures, analysis on mitochondria.

Validity type	Recommendation Category	Examples
Internal	Choice of sample size	Power calculation, larger samples sizes
	Randomized allocation of animals to treatment	Various methods of randomization
	Blinding of outcome assessment	Blinded measurement or analysis
	Flow of animals through an experiment	Recording animals excluded from treatment through to analysis
	Selection of appropriate control groups	Using negative, positive, concurrent, or vehicle control groups
	Study of dose-response relationships	Testing above and below optimal therapeutic dose
Construct	Characterization of animal properties at baseline	Characterizing inclusion/exclusion criteria, disease severity, age or sex
	Matching model to the human manifestation of the disease	Matching mechanism, chronicity or symptoms
	Treatment response along a mechanistic pathway	Characterizing pathway in terms of molecular biology, histology, physiology or behavior
	Matching outcome measures to clinical settings	Using functional or non-surrogate outcome measures
	Matching model to the age of patients in clinical settings	Using aged or juvenile animals
External	Replication in different models of the same disease	Different transgenic strains or lesion techniques
	Independent replication	Different investigators or research groups
	Replication in different species	Rodents and nonhuman primates
Research program	Inter-study standardization of experimental design	Coordination between independent research groups

Validity type	Recommendation Category	Studies	n (Percent of guidelines Citing)
Internal	Choice of sample size	Zhang et al. (21); Benova et al. (22); Simko et al. (25); Simko et al. (26); Stacchiotti et al. (27); Cheng et al. (30); Liu et al. (31); Liu et al. (33); Repova et al. (35); Drobnik et al. (36); Petrosillo et al. (38).	61.11%
	Randomized allocation of animals to treatment	Zhang et al. (21); Chen et al. (23); Simko et al. (25); Simko et al. (26); Salmanoglu et al. (29); Cheng et al. (30); Liu et al. (31); Liu et al. (33); Drobnik et al. (34); Repova et al. (35); Drobnik et al. (36).	61.11%
	Blinding of outcome assessment	Zhang et al. (21); Benova et al. (22); Chen et al. (23); Simko et al. (25); Liu et al. (24); Simko et al. (26); Stacchiotti et al. (27); Chaudagar et al. (28); Salmanoglu et al (29); Cheng et al. (30); Liu et al. (31); Zhu et al. (32); Liu et al. (33); Drobnik et al. (34); Repova et al. (35); Drobnik et al. (36); Chen et al. (37); Petrosillo et al. (38).	100%
	Flow of animals through an experiment	-	-
	Selection of appropriate control groups	Zhang et al. (21); Chen et al. (23); Simko et al. (25); Simko et al. (26); Stacchiotti et al. (27); Chaudagar et al. (28); Salmanoglu et al. (29); Cheng et al. (30); Liu et al. (31); Liu et al. (33); Drobnik et al. (34); Repova et al. (35); Drobnik et al. (36); Petrosillo et al. (38).	77.77%
	Study of dose-response relationships	Chen et al. (23); Simko et al. (25); Simko et al. (26); Chaudagar et al. (28); Cheng et al. (30); Liu et al. (31); Liu et al. (33); Drobnik et al. (34); Repova et al. (35); Drobnik et al. (36); Petrosillo et al. (38).	61.11%
Construct	Characterization of animal properties at baseline	Zhang et al (21); Benova et al. (22); Chen et al. (23); Simko et al. (25); Liu et al (24); Simko et al. (26); Stacchiotti et al. (27); Chaudagar et al. (28); Salmanoglu et al. (29); Cheng et al. (30); Liu et al. (31); Zhu et al. (32); Liu et al. (33); Drobnik et al. (34); Repova et al. (35); Drobnik et al. (36); Chen et al. (37); Petrosillo et al. (38).	100%
	Matching model to the human manifestation of the disease	Zhang et al. (21); Benova et al. (22); Chen et al. (23); Simko et al. (25); Simko et al. (26); Salmanoglu et al. (29); Cheng et al. (30); Liu et al. (31); Zhu et al. (32); Liu et al. (33); Drobnik et al. (34); Repova et al. (35); Drobnik et al. (36); Chen et al. (37); Petrosillo et al. (38).	88.88%
	Treatment response along a mechanistic pathway	Zhang et al. (21); Benova et al. (22); Chen et al. (23); Simko et al. (25); Liu et al. (24); Simko et al. (26); Stacchiotti et al. (27); Chaudagar et al. (28); Salmanoglu et al. (29); Cheng et al. (30); Liu et al (31); Zhu et al. (32); Liu et al. (33); Drobnik et al. (34); Repova et al. (35); Drobnik et al. (36); Chen et al. (37); Petrosillo et al. (38).	100%
	Matching outcome measures to clinical settings	Chen et al. (23); Simko et al. (25); Simko et al. (26); Stacchiotti et al. (27); Chaudagar et al. (28); Salmanoglu et al. (29); Cheng et al. (30); Liu et al. (31); Liu et al. (33); Repova et al. (35); Chen et al. (37); Petrosillo et al. (38).	66.66%
	Matching model to the age of patients in clinical settings	Zhang et al. (21); Benova et al. (22); Chen et al. (23); Simko et al. (25); Liu et al. (24); Simko et al. (26); Stacchiotti et al. (27); Chaudagar et al. (28); Salmanoglu et al. (29); Liu et al. (31); Zhu et al. (32); Liu et al. (33); Drobnik et al. (34); Repova et al. (35); Drobnik et al. (36); Chen et al. (37); Petrosillo et al. (38).	100%
External	Replication in different models of the same disease	-	-
	Independent replication	Zhang et al. (21); Chen et al. (23); Simko et al. (25); Simko et al. (26); Stacchiotti et al. (27); Chaudagar et al. (28); Salmanoglu et al. (29); Cheng et al. (30); Zhu et al. (32); Drobnik et al. (34); Repova et al. (35); Drobnik et al. (36); Chen et al. (37); Petrosillo et al. (38).	77.77%
	Replication in different species	Zhang et al. (21); Benova et al. (22); Chen et al. (23); Simko et al. (25); Simko et al. (26); Stacchiotti et al. (27); Chaudagar et al. (28); Salmanoglu et al. (29); Liu et al. (31); Zhu et al. (32); Liu et al. (33); Drobnik et al. (34); Repova et al. (35); Drobnik et al. (36); Chen et al. (37); Petrosillo et al. (38).	88.88%
Research program	Inter-study standardization of experimental design	Simko et al. (25); Simko et al. (26); Stacchiotti et al. (27); Chaudagar et al. (28); Zhu et al. (32); Repova et al. (35); Chen et al. (37).	38.88%

Authors	Assessments
Zhang et al. (21)	S	S	S	S	S	S
	Echocardiography measurements	Apoptosis analysis	Western blotting	Creatinine kinase measurement	Immunohistochemical analysis	Detection of autophagosomes
Benova et al. (22)	S	S	S	S	NS
	Biometric analysis	Western blotting	qRT-PCR	Hemodynamics measures
Chen et al. (23)	S	S	S	S	S	S
	Measurements of the infarct size	Measurement of lactate dehydrogenase	Measures of CMEC in vitro IRI assay	Western blotting	qRT-PCR	Detection of autophagosomes
Liu et al. (24)	S	S	S	S	S
	Echocardiography measurements	Hemodynamics measurements	Apoptosis analysis	Western blotting	qRT-PCR
Simko et al. (25)	S	S	NS	S	NS
	Biometric analysis	Hemodynamics measures	Determination of hydroxyproline	Angiotensin analysis	Aldosterone analysis
Simko et al. (26)	NS	S	S	S	S
	Hemodynamics measures	Determination of hydroxyproline	NO synthase activity	Oxidative load	Measurement and western blotting of NF-_ΚB
Stacchiotti et al. (27)	S	S	S	S
	Histomorphometrically evaluations	Nuclear cardiomyocyte morphometric	Mitochondrial analysis	Immunohistochemical analysis
Chaudagar et al. (28)	NS	S	S
	Hemodynamics measures	Biometric analysis	NO assays
Salmanoglu et al. (29)	NS	NS	S	S	NS
	Vasocontractile response	Measures of total cholesterol, LDL, HDL	NO assays	MDA assay	Measurements of tissue antioxidant levels
Cheng et al. (30)	S	S	S
	Immunohistochemical analysis	Western blotting	qRT-PCR
Liu et al. (31)	S	S	S
	Measurements of the infarcted size	Western blotting	Determination of the opening degree of MPTPs
Zhu et al. (32)	S	S	S
	Measurements of cell cultures antioxidant properties	Apoptosis analysis	LV functions
Liu et al. (33)	S	S	S	S
	Hemodynamics measures	Apoptosis analysis	Electron microscope examination	Analysis on mitochondria
Drobnik et al. (34)	S	S	S
	Determination of collagens	Determination of glycosaminoglycans	Electron microscope examination
Repova et al. (35)	S	S
	Hemodynamics measures	Determination of collagen
Drobnik et al. (36)	S	NS	S
	Estimation of lipid peroxidation	Determination of collagen	Determination of glycosaminoglycans
Chen et al. (37)	S	S	S	S	S
	Cardiac function	Hemodynamics measures	Lactate dehydrogenase	Apoptosis analysis	Immunohistochemistry
Petrosillo et al. (38)	S	S	S	S
	Measurements of the infarct size	Lactate dehydrogenase	Hemodynamics measures	Analysis on mitochondria

[1] *Corresponding author. E-mail: eduveiga56@gmail.com

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Melatonin and the cardiovascular system in animals: systematic review and meta-analysis