The Pd phosphor Pd(II) meso-tetra (sulfophenyl) tetrabenzoporphyrin sodium salt (C${}_{60}$H${}_{32}$N${}_4$Na${}_4$O${}_{12}$PdS${}_4$; catalog # T41161) was purchased from Frontier Scientific (Logan, UT, USA); it was dissolved in dH${}_2$O (2.5 mg/mL = 2 mM) and stored in small aliquots at –20 °C (single use). Potassium cyanide (1.0 M) was prepared daily in dH${}_2$O; the pH was adjusted to ~7.0 with 12 N HCl (added at an in vitro concentration of approximately 10 mM). Glucose oxidase (10 mg/mL) was dissolved in dH${}_2$O and stored at –20 °C. Sirolimus (rapamycin, molecular weight 914.172 g/mol; added at an in vitro concentration of 270 $\mu$M) was purchased from MedChem Express, LLC (Princeton, NJ); it was dissolved in dimethyl sulfoxide (DMSO) at 50 $\mu$g/$\mu$L (54.7 mM) and stored in small aliquots at –20 °C. Ozanimod (RPC1063; molecular weight 404.46 g/mol) was purchased from SelleckChem; it was dissolved at 0.06 mg/mL (136 $\mu$M) in ‘5% DMSO, 5% Tween-20, and 90% 0.1 N HCl’ and stored in small aliquots at –20 °C. The remaining reagents were purchased from Sigma-Aldrich (St. Louis, MO).

C57BL6 mice (typically, 8 to 16 weeks old) were housed at 22 °C with 60% humidity and 12-h light-dark cycles. Rodent chow and filtered water were provided ad libitum. Sodium pentobarbital (90 $\mu$g/g, given intraperitoneally) was used for anesthesia. The study was approved from the UAE University Animal Research Ethics Committee (Ref. ERA-2019-6026).

The thymus was quickly removed with a sterile scalpel and immersed in ice-cold phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na${}_2$HPO${}_4$, and 1.4 mM KH${}_2$PO${}_4$, pH 7.4), 3 $\mu$M Pd phosphor, and 0.5% fat-free albumin. A thymic fragment (typically, 10 to 20 mg of the outer cortex) was then quickly weighed and immediately placed in the oxygen phosphorescence analyzer (see below); ‘one thymic fragment per mouse per experimental condition’. Mixing was with the aid of parylene-coated stirring bars (Specialty Coating Systems Inc., Japan). Thymocyte respiration was determined at 25 °C in closed glass vials containing 2.0 mL PBS, 3 $\mu$M Pd phosphor, 0.5% fat-free albumin, and the tested drug or its diluent, as previously described [4, 5, 6, 7]. Experiments were performed with or without the addition of 5.0 mM glucose, as signified in Results. Since it was not possible to remove size-identical fragments, the rate of respiration was adjusted by the sample weight. Nevertheless, the same thymic region was targeted for all experiments.

Ozanimod was given by oral gavage at 0.6 mg/kg/day (0.57 $\mu$mol/kg/day) to 16-week-old female C57BL6 mice for 30 consecutive days, as previously described [13].

The previously described (home-made) phosphorescence oxygen analyzer was used to monitor thymocyte respiration [4, 5, 6, 7, 15, 16, 17, 18]. The principle of measurements was based on oxygen quenching the phosphoresce of the Pd phosphor. Briefly, the above listed Pd phosphor (absorption maximum, 625 nm; phosphorescence maximum, 800 nm) was used as an oxygen sensor. The decay rate (1/$\tau$) of the phosphorescence intensity (I) of this probe was set as: I = Ae${}^{-\text{𝑡}/\tau }$ [15]. The values of 1/$\tau$ were linear with dissolved oxygen concentration [O${}_2$]: 1/$\tau$ = $1/{\tau }^o$ + k${}_q$[O${}_2$], where 1/$\tau$ = phosphorescence decay rate in the presence of oxygen, $1/{\tau }^o$ = phosphorescence decay rate in the absence of oxygen, and k${}_q$ (s${}^{-1}$$\mu$M${}^{-1}$) = second-order oxygen quenching rate constant [15]. The measurements were performed in sealed glass vials exposed to flashes (600 per min) from a pulsed light-emitting diode array with a peak output at 625 nm. Emitted phosphorescence (at 800 nm) was detected by Hamamatsu photomultiplier tube (type 932). The amplified phosphorescence decay was then digitized at 1.0 MHz by a 20-MHz A/D converter, as previously described [17]. A program was developed using Microsoft Visual Basic 6, Microsoft Access Database 2007, and Universal Library components (Universal Library for Measurements Computing Devices; http://www.mccdaq.com/daq-software/universal-library.aspx). It allowed a direct reading from the PCI-DAS 4020/12 I/O Board (PCI-DAS 4020/12 I/O Board; http://www.mccdaq.com/pci-data-acquisition/PCI-DAS4020-12.aspx). Pulse detection was accomplished by searching for 10 phosphorescence intensities $>$1.0 volt. Peak detection was accomplished by searching for the highest 10 data points of a pulse and choosing the data point closest to the pulse decay curve [16]. Oxygen concentration decreased linearly with time, reflecting the zero-order kinetics of mitochondrial oxygen consumption. Thus, the respiration rate (k, in $\mu$M O${}_2$ min${}^{-1}$) was the negative of the slope d[O${}_2$]/dt. Cyanide inhibited respiration, confirming O${}_2$ was consumed in the respiratory chain by cytochrome oxidase. Dissolved oxygen in the measuring vials was rapidly depleted by the addition of glucose oxidase (catalyzes: D-glucose + O${}_2$$\to$ D-glucono-$\delta$-lactone + H${}_2$O${}_2$). The calibration reactions contained PBS with 3 $\mu$M Pd phosphor, 0.5% fat-free albumin, 50 $\mu$g/mL glucose oxidase, and various concentrations of $\beta$-glucose, as previously described [15].

It is worth noting that the high noise at the start of runs (air-saturated solutions) reflects the diminutive phosphorescence that reaches the photomultiplier tube (PMT), as dissolved oxygen quenches the light. By contrast, the low noise at the end of runs (oxygen-depleted solutions) reflects the augmentative phosphorescence that reaches the PMT. It is important, however, to realize that this phenomenon does not affect an accurate determination of the rate when a thymic fragment is present.

Data were analyzed on SPSS version 19 statistical package (SPSS Inc., New York, USA), using the nonparametric (2 independent samples) Wilcoxon test.

Fig. 1 shows a typical experiment involving thymocyte respiration. First, we establish the ‘drift rate’ in the reaction mixture (PBS + albumin + Pd phosphor) without thymic tissue. This ‘noise level’ was $\le$10% of the signal (Panel A). A newly collected thymic fragment (see Methods) was then added, and oxygen consumption was monitored as a function of time. As expected, the rate of thymocyte respiration slowed down, confirming the process was driven by the intracellular nutrients (Panel B, before the addition of glucose). At this point, the addition of glucose provided an external source of metabolic fuel, which established a steady-state rate of thymocyte respiration. These consecutive steps provided an insight into the thymocyte energy conversion (storage) pools. To avoid an irreversible injury from intracellular nutrient depletion (e.g., cell swelling as a result of ATP depletion), glucose must be added when the rate of respiration begins to slow down (see Panel B, after the addition of glucose). The addition of glucose oxidase tested the availability of dissolved oxygen and sets the numerical value of ‘zero oxygen concentration’ (Panel B). Respiration was inhibited by the addition of cyanide, confirming oxygen reduction occurred in cytochrome oxidase (Panel C).

Fig. 1.

Thymocyte respiration. Representative runs from five separate experiments are shown. Panel (A) shows the reaction mixture without thymic tissue (drift rate). In Panels (B–C), the reaction mixtures contained a fragment of the thymus. In Panel C, the reaction mixture also contained 1.0 mM potassium cyanide. Cyanide inhibited thymocyte respiration, confirming oxygen was consumed in the mitochondrial respiratory chain. Additions of glucose (10 mM) and glucose oxidase (0.1 mg/mL) are shown. Glucose oxidase catalyzes the reaction: D-glucose + O${}_2$$\to$ D-glucono-$\delta$-lactone + H${}_2$O${}_2$; its addition depleted dissolved oxygen in the solution. The rate of thymocyte respiration (k, $\mu$M O${}_2$ min${}^{-1}$) was the negative of d[O${}_2$]/dt. The values of k were corrected by the sample size and expressed as $\mu$M O${}_2$ min${}^{-1}$ mg${}^{-1}$ (k${}_c$). The lines are linear fits.

The overall rate of thymocyte respiration (mean $\pm$ SD, in $\mu$M O${}_2$ min${}^{-1}$ mg${}^{-1}$) was 0.046 $\pm$ 0.011 (median = 0.043, range = 0.028 to 0.062, n = 10). It is important to note the variability of thymocyte respiration, a limitation that prohibited establishing a valid reference range. Therefore, these experiments should be performed in littermates, or in at least age-matched mice. The drug experiments described below were performed in littermates.

Runs similar to the above were then performed in the presence of 270 $\mu$M sirolimus or 10 $\mu$L of the vehicle DMSO. As shown in Fig. 2, the rate of thymocyte respiration (k${}_c$, in $\mu$M O${}_2$ min${}^{-1}$ mg${}^{-1}$) with DMSO was 0.067 and with sirolimus was 0.050 (25% lower). The effect of sirolimus was notable after approximately 60 min. Overall, the values of k${}_c$ (mean $\pm$ SD, in $\mu$M O${}_2$ min${}^{-1}$ mg${}^{-1}$) with DMSO was 0.063 $\pm$ 0.009 (median = 0.064, range = 0.040 to 0.078, n = 12) and with sirolimus was 0.058 $\pm$ 0.015 (median = 0.054 [16% lower, p = 0.4248], range = 0.042 to 0.077, n = 6). Similarly, dose-response curves for sirolimus with or without the addition of ‘response modifier(s)’ could be similarly investigated.

Fig. 2.

In vitro effects of sirolimus on thymocyte respiration. The experimental conditions were as described in the legend to Fig. 1. Representative runs from at least six separate experiments are shown. The lines are linear fits. The rate of thymocyte respiration ($\mu$M O${}_2$ min${}^{-1}$ mg${}^{-1}$) with 10 $\mu$L DMSO was 0.067 and with 270 $\mu$M sirolimus was 0.050 (25% lower).

Fig. 3 shows an inhibitory effect of ozanimod on thymocyte respiration, similar to that of sirolimus. The runs were in the presence of 0.68 $\mu$M ozanimod or 10 $\mu$L diluent (5% DMSO, 5% Tween-20, and 90% 0.1 N HCl). The rate of thymocyte respiration (in $\mu$M O${}_2$ min${}^{-1}$ mg${}^{-1}$) with the diluent was 0.059 and with ozanimod was 0.039 (34% lower). Overall, the values of k${}_c$ ($\mu$M O${}_2$ min${}^{-1}$ mg${}^{-1}$) in the presence of the diluent was 0.055 $\pm$ 0.011 (median = 0.059, range = 0.040 to 0.062, n = 3) and in the presence of ozanimod was 0.045 $\pm$ 0.009 (median = 0.042 [29% lower, p = 0.4], range = 0.039 to 0.055, n = 3).

Fig. 3.

In vitro effects of ozanimod on thymocyte respiration. The experimental conditions were as described in the legend to Fig. 1. Representative runs from three separate experiments are shown. The rate of thymocyte respiration ($\mu$M O${}_2$ min${}^{-1}$ mg${}^{-1}$) with the addition of 10 $\mu$L diluent was 0.059 and with the addition of 0.68 $\mu$M ozanimod was 0.039 (34% lower).

We then examined the effects of an in vivo treatment with ozanimod (0.6 mg/kg/day, or 0.57 $\mu$mol/kg/day) on thymocyte respiration at the completion of 30-consecutive days of administration. As shown in Fig. 4A, this treatment caused weight loss. Nevertheless, thymocyte respiration was similar in mice given ozanimod compared to those given the diluent (Fig. 4B). Overall, the values of k${}_c$ ($\mu$M O${}_2$ min${}^{-1}$ mg${}^{-1}$) in the presence of the diluent was 0.041 $\pm$ 0.010 (median = 0.041, range = 0.026 to 0.056, n = 7) and in the presence of ozanimod was 0.047 $\pm$ 0.016 (median = 0.043, range = 0.028 to 0.079, n = 8; p = 0.6), Fig. 4C.

Fig. 4.

In vivo effects of ozanimod on thymocyte respiration. C57BL6 female mice (16-week-old) were given daily ozanimod (0.6 mg/kg, or 1.36 mmol/kg) by oral gavage for 30 consecutive days. Control mice received the diluent (10 $\mu$L/g of body weight of ‘5% DMSO, 5% Tween 20, and 90% of 0.1N HCl’). Two separate experiments, each had four mice per group. Panel (A) shows the relative weight change with time (a representative of two separate experiments is shown). Asterisk: One ozanimod-treated mouse died on Day 15 after losing about 29% of the body weight. Panel (B): Representative runs from four separate experiments of thymocyte respiration following the 30-day treatment with ozanimod or diluent. The experimental conditions were as described in the legend to Fig. 1. Panel (C), dot plot summarizing the rats of respiration.