Introduction

Most mammals display marked annual cycles of body fattening and energy metabolism as a strategy to promote winter survival and reproductive success. In sheep, photoperiodism, the physiological response of organisms to the ratio of the day to the night length, represents a developmental strategy of conservation having a direct impact on reproduction by alternating periods of sexual rest and periods of sexual activity. These natural cycles regulate appetite and energy expenditure and represent an interesting way for a better understanding of the hypothalamic mechanisms of metabolic control. The objective of this study was to examine the impact of photoperiodism on the hypothalamic metabolism in female sheep using proton MR Spectroscopy.

Materials and Methods

A cohort of 8 female sheep were examined in a dedicated Siemens MRI scanner (Siemens, Verio, Erlangen, Germany) at a field strength of 3T at 4 time points (P01, P02, P03 and P04) from May till July during long days (LD) and at 4 time points from late September till November during short days (SD). 2 weeks were allowed between subsequent time points. Weekly blood samplings were performed from March till November to follow their hormonal status. Prior to MRI, each sheep was fasted overnight and intubated under ketamine and xylazine anaesthesia (20 mg/kg). Each ewe was then transported to the MRI room, installed prone on the MRI bed and anaesthesia was immediately switched to 2% Isoflurane in medical air through a respirator (Aestiva, GE Healthcare, Datex-Ohmeda, USA). The respirator allowed continuous control of respiration rates. An oximeter was attached to one of the hind-paws allowing for the control of the partial pressure of oxygen and heart rate. The temperature was controlled through MRI-compatible rectal probe. Structural images were acquired using the T1-weighted 3D magnetization prepared rapid gradient echo (MPRAGE) sequence (TR/TE/TI=2500/318/900 ms ; Flip angle = 12; NEX= 2 ; FOV=192 x 192 mm2, Matrix size = 384 x384; Voxel size = 0.5 x 0.5 x 0.5 mm3). MR spectroscopy was conducted using a STEAM sequence (TR/TE/TM= 3000/5/30 ms ; 256 acquisitions) in a large voxel of interest (VOI = 10 x 12 x 13 mm3) covering the entire hypothalamus. FASTESTMAP was used for the shim down to a water linewidth of 10 ± 2Hz. Collected MRS data were analyzed using LCModel (1) and absolute metabolite quantification was obtained using the unsuppressed water signal acquired in the same VOI. A two-way ANOVA with Bonferroni correction was used for statistical analysis. p<0.05 was considered significant.

Results

Figure 1 depicts a T1-weighted MPRAGE image with the position of the VOI within the hypothalamus (A) as well as a representative example of proton MR spectrum acquired within this VOI (B) at 3T. Respective LCModel fitted spectra acquired during LD (C) and SD (D) are also shown. Figure 2 A-D present comparisons of neurochemical profiles (NPs) at 4 time points during LD and SD. Significant differences were found at P01 with decreased glutamine (Gln) (p = 0.01) and glutamate (Glu) (p= 0.039) as well as myo-Inositol (mI), NAA and Glu + Gln (p< 0.03) during SD compared to LD. At P02, no significant differences were found. Only NAA levels were significantly different at P03 while significant increases of Gln, Glu, NAA and Glu + Gln (p < 0.03) were measured at P04 during SD compared to LD. Figure 3 displays NPs at 4 time points during LD and SD respectively. Creatine (Cr), N-acetyl-Aspartate (NAA) and mI levels were significantly higher at P01 versus P04 and Glu at P03 versus P02 and P04 during LD (p < 0.001). During SD, Gln and Glu were significantly lower at P01 compared to all other time points. During LD, glucose (Glc) levels demonstrated a tendency to increase as a function of time without significant difference (p> 0.05).

Discussion

Photoperiodic regulation of metabolism has already been reported in the past in the hypothalamus of other species (2). To the best of our knowledge, 1H-MRS was not used to examine the impact of photoperiodism in the sheep brain. Preliminary results confirm significant differences of the hypothalamic metabolism between LD and SD as reported in the past (2. Notably, decreased Gln and Glu levels during SD were already shown and attributed to a reduced transport by tanycytes involved in the transport of a amino acids from brain capillaries and likely involved in the regulation of transport across the endothelial layer. Our present results also show that metabolite concentration changes were transient during SD and more pronounced during LD. Our data suggest that photoperiodism in large brain animals such sheep may be useful for a better understanding of the hypothalamic environment.

Acknowledgements

This study was funded by a grant from Agence Nationale de la Recherche (ANR-16-CE37-0006-01)