Evaluation of hematology, general serum biochemistry, bone turnover markers and bone marrow cytology in a glucocorticoid treated ovariectomized sheep model for osteoporosis research

COELHO, CATARINA A.; BORDELO, JOÃO P.; CAMASSA, JOSÉ A.; BARROS, VERA A.; BABO, PEDRO S.; GOMES, MANUELA E.; REIS, RUI L.; AZEVEDO, JORGE T. DE; REQUICHA, JOÃO F.; FAÍSCA, PEDRO; CARVALHO, PEDRO P.; VIEGAS, CARLOS A.; DIAS, ISABEL R.

doi:10.1590/0001-3765202020200435

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BIOMEDICAL SCIENCES • An. Acad. Bras. Ciênc. 92 (4) • 2020 • https://doi.org/10.1590/0001-3765202020200435 copy

Evaluation of hematology, general serum biochemistry, bone turnover markers and bone marrow cytology in a glucocorticoid treated ovariectomized sheep model for osteoporosis research

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Osteoporosis is a metabolic disorder characterized by a loss of bone mass and structure and increasing the risk of fragility fractures, mostly among postmenopausal women. Sheep is a recognized large animal model for osteoporosis research. An experimental group of ewes (3-4 years old) was subjected to ovariectomy (OVX) and weekly glucocorticoid (GC) application for 24 weeks and compared with a sham control group. Blood and bone marrow parameters were analyzed before and 24 weeks after OVX and GC administration. Osteopenia was confirmed through micro-computed tomography and histomorphometric analysis of L4 vertebra in the study end. A statistically significant increase was observed in mean corpuscular volume, mean cell hemoglobin and monocytes and a decrease in red blood count and eosinophils (p<0.05). Alkaline phosphatase (ALP), gamma-glutamyl transpeptidase, magnesium and α1-globulin increased, and creatinine, albumin, sodium and estradiol decreased (p<0.05). A slight decrease of bone formation markers (bone ALP and osteocalcin) and an increase of bone resorption markers (C-terminal telopeptides of collagen type 1 and tartrate-resistant acid phosphatase) were observed, but without statistical significance. This study aims to contribute to better knowledge of sheep as a model for osteoporosis research and the consequences that a performed induction protocol may impose on organic metabolism.

Key words
animal model; blood analysis; bone marrow cytology; lumbar vertebral microstructure; osteoporosis; sheep

Hematological parameters	GC-treated OVX sheep group		Ovine species reference range
Hematological parameters	Before	After	Ovine species reference range
Red blood cell parameters
Erythrocytes (x10⁶/µL)	9.12	8.65*	9-15
Hemoglobin (g/dL)	10.9	10.9	9-15
Hematocrit (%)	40.4	42.3	27-45
Mean corpuscular volume (fL)	39.7	47.9*	28-40
Mean cell hemoglobin (pg)	11.1	12.5*	8-12
Mean corpuscular hemoglobin concentration (%)	28.0	26.3	31-34
White blood cell parameters
Leucocytes (x10³/µL)	7.32	11.11	4.0-12.0
Neutrophils (band cells) (x10³/µL)	-	-	rare
Neutrophils (polymorphonuclear cells) (x10³/µL)	2.53	7.92	0.7-6.0
Lymphocytes (x10³/µL)	4.16	2.92	2.0-9.0
Monocytes (x10³/µL)	0.21	0.55*	0.0-0.75
Eosinophils (x10³/µL)	0.31	0.02*	0.0-1.0
Basophils (x10³/µL)	0.02	0.02	0.0-0.3
Other parameters
Total platelet count (x10³/µL)	352	410	800-1100 (300-800)

	GC-treated OVX sheep group		Ovine species reference range
Serum biochemical parameters	Before	After	Ovine species reference range
Metabolites
Glucose (mmol/L)	3.28	2.89	2.78-4.45
Total cholesterol (mmol/L)	1.62	1.39	1.11-2.66
Renal function
Blood nitrogen urea (mmol/L)	5.0	7.5	1.67-5.83
Creatinine (µmol/L)	73.4	59.2*	106-168
Enzymes
Total alkaline phosphatase (U/L)	52	98*	70-390
Aspartate aminotransferase (U/L)	132	133	60-280
Alanine aminotransferase (U/L)	31	24	22-38
Gamma-glutamyl transferase (U/L)	56	81*	20-52
Minerals
Total calcium (mmol/L)	2.33	2.1	2.88-3.25
Phosphorus (mmol/L)	1.84	2.16	1.6-2.4
Magnesium (mmol/L)	0.84	1.0*	0.90-1.15
Proteins
Total protein (g/dL)	7.5	6.4	6-7.9
Albumin (g/dL)	3.64	3.15*	2.4-3
Alpha 1-globulin (g/dL)	0.34	0.37*	-
Alpha 2-globulin (g/dL)	0.81	0.81	-
Beta-globulin (g/dL)	0.40	0.53	-
Gamma-globulin (g/dL)	2.44	1.97	-
Electrolytes
Sodium (mmol/L)	151	149*	145-152
Potassium (mmol/L)	4.6	4.9	3.9-5.4
Chloride (mmol/L)	110	102	95-103
Bone turnover markers
Bone-specific alkaline phosphatase (U/L)	7.06	8.5	5.8-25.8
Intact osteocalcin (µg/L)	9.54	4.89	9.3-16
C-terminal telopeptides of collagen type 1 (pg/L)	0.31	0.52	0.2-0.45
Tartrate-resistant acid phosphatase (U/L)	2.1	2.5	0.14-5.9
Hormones
Estradiol (pmol/L)	183.5	139.5*	76.3 (mean) (29-117)

Bone marrow composition	GC-treated OVX sheep group
Cell type	Before	After
Hematopoietic cells – Myeloid series
Myeloblasts and promyelocytes (%)	3.0	3.1
Myelocytes, metamyelocytes and segmented granulocytes (%)	32.0	31.7
Eosinophils (%)	6.2	7.5
Basophils (%)	0.0	0.0
Total myeloid series (%)	41.2	42.3
Hematopoietic cells – Erythroid series
Rubriblasts and prorubricytes (%)	2.0	3.55
Rubricytes and metarubricytes (%)	54.3	52.9
Total erythroid series (%)	56.3	56.4
Myeloid/erythroid ratio (%)	0.76	0.75
Lymphocytes (Lymph) (%)	1.7	0.65
Plasma cells (%)	0.5	0.18
Monocytes (%)	0.0	0.35
Megakaryocytes (%)	0.5	0.55

	L4 vertebral body
	Sham control group	GC-treated OVX sheep group
µCT parameters
BV/TV (%)	44.1	42.5
BS/BV (1/mm)	18.1	18.6
Tb.Th (mm)	0.13	0.14
Tb.Sp (mm)	0.35	0.40
Tb.N (1/mm)	3.39	3.05
Po(cl) (%)	0.20	0.15
Po(op) (%)	52.7	59.4
Po(tot) (%)	52.9	59.5
BMD (g/cm³)	0.69	0.62
Histomorphometric parameters
Ct.Th (µm)	693.8	721.4
Ct.Po (%)	5.0	8.8
BV/TV (%)	43.2	34.7
Tb.Th (µm)	218.2	174.8
Tb.Sp (µm)	366.7	430.8*
Tb.N (#/mm)	3.7	3.2

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[1] Correspondence to: Isabel R. Dias E-mail: idias@utad.pt *Authors that equally contributed to the study