Effects of Citalopram on Sutural and Calvarial Cell Processes

Abstract

The use of selective serotonin reuptake inhibitors (SSRIs) for the treatment of depression during pregnancy is suggested to increase the incidence of craniofacial abnormalities including craniosynostosis. Little is known about this mechanism, however based on previous data we propose a mechanism that affects cell cycle. Excessive proliferation, and reduction in apoptosis may lead to hyperplasia within the suture that may allow for differentiation, bony infiltration, and fusion. Here we utilized in vivo and in vitro analysis to investigate this proposed phenomenon. For in vivo analysis we used C57BL-6 wild-type breeders treated with a clinical dose of citalopram during the third trimester of pregnancy to produce litters exposed to the SSRI citalopram in utero. At post-natal day 15 sutures were harvested from resulting pups and subjected to histomorphometric analysis for proliferation (PCNA) and apoptosis (TUNEL). For in vitro studies, we used mouse calvarial pre-osteoblast cells (MC3T3-E1) to assess proliferation (MTS), apoptosis (Caspase 3/7-activity), and gene expression after exposure to titrated doses of citalopram. In vivo analysis for PCNA suggested segregation of effect by location, with the sagittal suture, showing a statistically significant increase in proliferative response. The coronal suture was not similarly affected, however there was a decrease in apoptotic activity at the dural edge as compared to the periosteal edge. No differences in apoptosis by suture or area due to SSRI exposure were observed. In vitro results suggest citalopram exposure increased proliferation and proliferative gene expression, and decreased apoptosis of the MC3T3-E1 cells. Decreased apoptosis was not confirmed in vivo however, an increase in proliferation without a concomitant increase in apoptosis is still defined as hyperplasia. Thus prenatal SSRI exposure may exert a negative effect on post-natal growth through a hyperplasia effect at the cranial growth sites perhaps leading to clinically significant craniofacial abnormalities.

Fig 1. Histological markers of proliferation in control and citalopram exposed mouse cranial sutures.

A & B. Coronal sutures from control (A) and citalopram exposed (B) 15 day old C57BL6 mice stained for Proliferating Cell Nuclear Antigen (PCNA) (black arrows = example of positive cell, white arrows = example of negative cells). Periosteum, dura and osteogenic front (OF) are as marked, and the sutural area has been outlined. C. Quantification of percent PCNA stain. PCNA activity shows slight decreases for all but dural associated areas, but no statistical differences. D & E. Sagittal sutures from control (D) and exposed (E) 15 day old C57BL6 mice stained for PCNA. F. Quantification of percent PCNA stain. Overall, PCNA activity increased in all areas assessed in citalopram exposed sagittal sutures.

Fig 2. Assessment of in vitro markers of proliferation after citalopram treatment.

A. MTS functional assay results from MC3T3 cells treated with serial dilutions of citalopram (10⁻⁴ mol through 10⁻¹⁰ mol). Significant time and dose effects are noted. B. Gene expression markers of proliferation from MC3T3 cells treated with media only or media supplemented with 10⁻⁴ mol citalopram. All three markers indicate a biological change due to treatment by 7 days. Jun was found to have significantly greater expression due to treatment at both 3 and 7 days.

Fig 3. Histological markers of apoptosis in control and citalopram exposed mouse cranial sutures.

A & B. Coronal sutures from control (A) and citalopram exposed (B) 15 day old C57BL6 mice stained for Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) activity (green). Periosteum, dura and osteogenic front (OF) are as marked, and the sutural area has been outlined. Inset lower magnification of TUNEL staining with a 4',6-diamidino-2-phenylindole (DAPI) nuclear counterstain (blue). Note the highly cellularized (blue stained) area of the suture abutting the less cellularized (darker) area of the osteogenic front. C. Quantification of percent TUNEL positive staining as compared to DAPI stain in coronal sutures. Significant decreases in TUNEL activity were noted due to citalopram exposure at the periosteal and dural areas of the sutures. Midsutral space and osteogenic fronts did not demonstrate significantly different staining between unexposed and citalopram exposed sutures. D & E. Sagittal sutures from unexposed (control) (D) and citalopram exposed (E) 15 day old C57BL6 mice stained for TUNEL activity (green). Inset lower magnification of TUNEL staining with a DAPI nuclear counterstain (blue) F. Quantification of percent TUNEL positive staining as compared to DAPI stain. A significant increase in TUNEL staining was noted in the periosteal area while a significant decrease in staining was noted at the osteogenic front of citalopram exposed sagittal sutures.

Fig 4. Assessment of in vitro markers of apoptosis after citalopram treatment.

A. Caspase 3/7 functional assay results from MC3T3 cells treated with serial dilutions of citalopram (10⁻⁴ mol through 10⁻¹⁰ mol). No significantly significant differences were found by day, dose, or interaction terms. B. Gene expression markers of apoptosis from MC3T3 cells treated with media only or media supplemented with 10⁻⁴ mol citalopram. There were no statistically significant gene expression changes.