Ventilatory impairment in the dysmyelinated Long Evans shaker rat

Abstract

Although respiratory complications significantly contribute to morbidity/mortality in advanced myelin disorders, little is known concerning mechanisms whereby dysmyelination impairs ventilation, or how patients compensate (i.e. plasticity). To establish a model for studies concerning mechanisms of ventilatory impairment/compensation, we tested the hypotheses that respiratory function progressively declines in a model of CNS dysmyelination, the Long Evans shaker rat (les). The observed impairment is associated with abnormal inspiratory neural output. Minimal myelin staining was found throughout the CNS of les rats, including the brainstem and cervical bulbospinal tracts. Ventilation (via whole-body plethysmography) and phrenic motor output were assessed in les and wild-type (WT) rats during baseline, hypoxia (11% O(2)) and hypercapnia (7% CO(2)). Hypercapnic ventilatory responses were similar in young adult les and WT rats (2 months old); in hypoxia, rats exhibited seizure-like activity with sustained apneas. However, 5-6 month old les rats exhibited decreased breathing frequencies, mean inspiratory flow (V(T)/T(I)) and ventilation (V (E)) during baseline and hypercapnia. Although phrenic motor output exhibited normal burst frequency and amplitude in 5-6 month old les rats, intra-burst activity was abnormal. In WT rats, phrenic activity was progressive and augmenting; in les rats, phrenic activity was decrementing with asynchronized, multipeaked activity. Thus, although ventilatory capacity is maintained in young, dysmyelinated rats, ventilatory impairment develops with age, possibly through discoordination in respiratory motor output. This study is the first reporting age-related breathing abnormalities in a rodent dysmyelination model, and provides the foundation for mechanistic studies of respiratory insufficiency and therapeutic interventions.

Figure 1

Myelin staining (20X magnification) of the brainstem (A, B) and spinal cord segment C4 (C, D) taken from a 5–6 month old wild-type (WT, left panels) and mutant (les, right panels) rat. Insets are taken at 200X magnification. A sagittal section taken from the caudal ventro-lateral brainstem shows prominent myelin labeling in the WT rat (A) with no detectable myelin staining in the les rat (B). These brainstem areas contain respiratory neurons (e.g.. ventral respiratory column). Coronal sections of the cervical spinal cord segment C4 also confirm the absence of myelin in the les rat (D) whereas axons from the WT rat are heavily myelinated (C). Note that the peripheral motor nerve of the les rat remains myelinated (dark arrow, D). These segments include descending bulbospinal pathways to inspiratory motor neurons, associated with the phrenic nerve/diaphragm.

Figure 2

Baseline ventilation (normocapnia) and hypercapnic (FICO₂ = 7%) ventilatory responses in 2 month old WT (open circles, ○) and les (closed circles, ●) rats. In hypercapnia, WT and les rats exhibited similar changes in ventilation from baseline including increased breathing frequency, VT, V̇E, and VT/TI with corresponding decreases in TI and TE (RM ANOVA, *p < 0.05 from baseline; p > 0.05 between groups; A-F). However, les rats had significantly lower TE at baseline, resulting in an overall interaction between treatment and rat group (RM ANOVA, #p < 0.05 between rat groups, †p < 0.05 interaction; E).

Figure 3

Representative breathing traces in 5–6 month old WT (A, C) and les rats (B, D) during baseline and hypercapnia (FICO₂ = 7%). Breathing frequency is slower, with longer values of TI and TE at baseline and during hypercapnia in les rats (B, D). However, tidal volume (normalized to body mass) do not differ between groups. Summary data are shown in Figure 4. Figure 3E shows a young (2 month old) les rat during hypoxia (FIO₂ = 11%). The left side of the trace shows a normal breathing pattern with mild disturbances in the otherwise steady respiratory pattern due to rat movements. Dark arrow indicates seizure-like activity in the les rat followed by a prolonged period of apnea. Rats were unable to recover from these apneic periods and subsequently died unless rescued with ventilatory support and returned to normoxia.

Figure 4

Baseline ventilation (normocapnia) and hypercapnic (FICO₂ = 7%) ventilatory responses in 5–6 month old WT (open circles, ○) and les (closed circles, ●) rats. Similar to 2 month old rats, hypercapnia increased breathing frequency, VT, V̇E, and VT/TI with corresponding decreases in TI and TE (RM ANOVA, *p < 0.05 from baseline; p > 0.05 between groups; A-F). However, les rats had significantly slower breathing frequencies, with prolonged TI and TE, at baseline and during hypercapnia versus WT rats; VT did not differ between groups (RM ANOVA, #p < 0.05 between rat groups, A-C, E). V̇E and VT/TI were diminished during hypercapnia in les rats due to differences in timing variables (RM ANOVA, #p < 0.05 between rat groups, D, F); statistically significant interactions between group and treatment were found in TI and V̇E (RM ANOVA, †p < 0.05 interaction, C, D). Thus, ventilatory responses to hypercapnia are impaired in les rats when compared to WT rats at 5–6 months of age. When not apparent, error bars are within data points.

Figure 5

Expanded phrenic neurograms of an individual burst (breath) from a 5–6 month old WT (top) and les (bottom) rat during baseline. WT rats show a typical, incrementing neural discharge with a single peak. In contrast, les rats frequently displayed multi-peaked bursts with incrementing/decrementing neural activity and low instantaneous voltages.

Figure 6

Phrenic burst activity during hypercapnia in 5–6 month old WT (open circles, ○) and les (closed circles, ●) rats. Hypercapnia increased phrenic burst amplitude, minute phrenic activity and amplitude/TI, while decreasing TI to a similar extent in both les and WT rat groups (RM ANOVA, *p < 0.05 from baseline; p > 0.05 between groups; B, C-F). Phrenic burst frequency increased overall in hypercapnia (RM ANOVA, *p < 0.05 from baseline; A). Les rats had significantly shorter TI compared to WT rats at baseline, but not during hypercapnia (RM ANOVA, #p < 0.05 between rat groups, C). Thus, although unanesthetized hypercapnic ventilatory responses are impaired in les rats, hypercapnic phrenic responses are not.

Figure 7

Phrenic burst activity and mean arterial blood pressure during hypoxia (FIO₂ = 11%) in 5–6 month old WT (open circles, ○) and les (closed circles, ●) rats. Hypoxia significantly increased phrenic burst frequency, amplitude and minute activity to a similar extent in both les and WT rats (RM ANOVA, *p < 0.05 from baseline; p > 0.05 between groups; A-C). In contrast, overall mean arterial pressure significantly decreased during hypoxia, primarily as a result of a profound decrease in les rats (RM ANOVA, *p < 0.05 from baseline; #p < 0.05 between rat groups; D).