Neurosteroid and benzodiazepine combination therapy reduces status epilepticus and long-term effects of whole-body sarin exposure in rats

Abstract

Objective: Our objective was to evaluate the protective efficacy of the neurosteroid pregnanolone (3α-hydroxy-5β pregnan-20-one), a GABA_A receptor-positive allosteric modulator, as an adjunct to benzodiazepine therapy against the chemical warfare nerve agent (CWNA) sarin (GB), using whole-body exposure, an operationally relevant route of exposure to volatile GB.

Methods: Rats implanted with telemetry transmitters for the continuous measurement of cortical electroencephalographic (EEG) activity were exposed for 60 minutes to 3.0 LCt₅₀ of GB via whole-body exposure. At the onset of toxic signs, rats were administered an intramuscular injection of atropine sulfate (2 mg/kg) and the oxime HI-6 (93.6 mg/kg) to increase survival rate and, 30 minutes after seizure onset, treated subcutaneously with diazepam (10 mg/kg) and intravenously with pregnanolone (4 mg/kg) or vehicle. Animals were evaluated for GB-induced status epilepticus (SE), spontaneous recurrent seizures (SRS), impairment in spatial memory acquisition, and brain pathology, and treatment groups were compared.

Results: Delayed dual therapy with pregnanolone and diazepam reduced time in SE in GB-exposed rats compared to those treated with delayed diazepam monotherapy. The combination therapy of pregnanolone with diazepam also prevented impairment in the Morris water maze and reduced the neuronal loss and neuronal degeneration, evaluated at one and three months after exposure.

Significance: Neurosteroid administration as an adjunct to benzodiazepine therapy offers an effective means to treat benzodiazepine-refractory SE, such as occurs following delayed treatment of GB exposure. This study is the first to present data on the efficacy of delayed pregnanolone and diazepam dual therapy in reducing seizure activity, performance deficits and brain pathology following an operationally relevant route of exposure to GB and supports the use of a neurosteroid as an adjunct to standard anticonvulsant therapy for the treatment of CWNA-induced SE.

Keywords: benzodiazepine; chemical warfare nerve agent; neurosteroids; refractory status epilepticus; thalamus.

Figure 1

Whole‐body exposure chamber is shown on the left image, and a close‐up of setup inside the chamber (red circle) is shown on the right image. The chamber is equipped with telemetry receivers covered in foil outside the chamber and a plastic‐perforated enclosure inside the chamber to hold the rat. Access ports enabled treatment of animals via a catheter while in the chamber, as well as drawing of blood for analysis of agent levels in blood

Figure 2

Representative EEG tracings depicting the progression of EEG graphic activity before, during and after GB exposure and treatment (TR). A, The overview image corresponds to compressed EEG for a 24 h period. Note that GB‐exposed rats that received diazepam and pregnanolone (GB/DZP/PREG) 30 minutes after seizure onset had reduced overall amplitude of the signal faster than those that received diazepam monotherapy (GB/DZP). B, A 10‐s representative image of baseline EEG is shown. C, Ten min after SE onset, both groups presented high‐amplitude hypersynchronous epileptiform discharges. D, Five minutes after treatment, only the GB/DZP/PREG group showed total blockade of seizures. E, Twenty‐four hours after treatment the GB/DZP group still displayed high‐amplitude spikes, while the GB/DZP/PREG group had EEG similar to the baseline

Figure 3

Rats exposed via whole‐body inhalation to GB and treated with atropine sulfate and HI‐6 at onset of toxic signs and with diazepam with or without pregnanolone 30 min after seizure onset were evaluated for initial seizure duration and EEG power. A, Dual therapy with diazepam and pregnanolone (GB/DZP/PREG; n = 16) reduced initial seizure duration, terminating seizure activity within a few minutes of treatment, compared to rats treated with diazepam monotherapy (GB/DZP; n = 15) which displayed seizure activity for several hours after exposure (P < 0.001). B and C, The percent of relative change from baseline in the power of delta (0.1‐4 Hz) band was calculated for each group up to 4 and 24 h for GB/DZP/PREG and GB/DZP groups compared with values of control (Air control/No GB; n = 10) rats. The power spectra in the delta band returned to normal levels faster in the GB/DZP/PREG group when compared to GB/DZP group. During the refractory seizures period, GB/DZP power spectra remained elevated for sustained periods of time when compared with Air control/No GB, while the GB/DZP/PREG power spectra remained elevated only for a limited time (up to 8 h) when compared to Air control/No GB. Statistically significant differences between groups at specific time points are marked by a line indicated above or below the graphed data. *P < 0.05, GB/DZP compared with Air control/No GB. #P < 0.05, GB/DZP/PREG compared with Air control/No GB

Figure 4

Rats exposed to GB and treated with atropine sulfate and HI‐6 at onset of toxic signs and with diazepam monotherapy (GB/DZP; n = 7) 30 min after seizure onset lost significant weight by 24 h after exposure. Shown are body weight changes in rats that were evaluated daily (M‐F) over a 90‐d period after GB exposure. Body weight remained less than baseline and less than air control (Air control/No GB; n = 6) on post‐exposure days (PED) 1 and 2 (**P < 0.01). In contrast, rats exposed to GB that received pregnanolone and diazepam dual therapy (GB/DZP/PREG; n = 8) were not different from Air control/No GB initially after exposure, had significant gain above their baseline by PED 5 and by 2 mo after exposure weighed more than air control rats (P < 0.05). GB‐exposed rats tended to gain more weight than air control by 3 mo after exposure but this did not reach significance. B, Rats that received diazepam and pregnanolone dual therapy (n = 16) 30 min after GB‐induced seizure onset had greater reduction in body temperature between 1 and 6 h after exposure compared to air control rats treated with diazepam (n = 15; *P < 0.05). All groups had reduced body temperature compared with their baseline, with air control having reduced temperature 1‐4 h after air exposure, GB/DZP rats having reduced body temperature 2‐7 h after exposure and GB/DZP/PREG reducing temperature 1‐8 h after exposure

Figure 5

Rats exposed to GB and treated with atropine sulfate and HI‐6 at onset of toxic signs and with diazepam monotherapy (GB/DZP; n = 8) 30 min after seizure onset had impaired performance in Morris water maze test 1 mo after exposure, compared with Air control/No GB (n = 8) shown by (A) increased latency to escape, (B) increased path length (distance travelled), and (C) increased thigmotaxis (perimeter swim). Rats exposed to GB that received pregnanolone and diazepam dual therapy (GB/DZP/PREG; n = 15) were not impaired. All groups improved performance with repeated trials, but the diazepam‐treated GB‐exposed did not perform as well as the air control rats. D, There was no significant effect of GB on swim speed in any group, although all rats had greater swim speed on their first session (S1) compared with their other sessions. Re‐evaluation at 3 mo in a subset of rats showed that the GB/DZP group (n = 6) continued to show greater latency to escape and more thigmotaxis compared with Air control/NoGB (n = 5), while GB/DZP/PREG rats (n = 8) were not impaired. *P < 0.05; +P < 0.05

Figure 6

Silver stain of neuronal degeneration in brains was evaluated in rats euthanized 1 or 3 mo after whole‐body exposure to GB followed by atropine sulfate and HI‐6 at onset of toxic signs and diazepam with or without pregnanolone 30 min after seizure onset. Representative images of silver‐stained coronal sections from (A) Air control/No GB rats, (B) rats exposed to GB and treated with delayed diazepam monotherapy (GB/DZP), and (C) rats exposed to GB and treated with delayed diazepam and pregnanolone dual therapy (GB/DZP/PREG) showing neuronal fiber degeneration with darkened regions indicative of fiber and neuronal degeneration. D and E, A semi‐qualitative assessment of silver‐stained brain sections was performed, and the median neuropathology score for each group is presented in a box‐and‐whiskers graph (box: median or 50th percentile represented by bold line, with limits of box representing the 25th and 75th percentiles; whiskers: 10th and 90th percentiles). GB‐exposed rats that received diazepam had extensive neuropathology in the fiber tracts, thalamus and piriform cortex 1 mo after GB exposure (D), as well as in the amygdala 3 mo (E) after GB exposure compared with air control rats (+P < 0.05). Rats that received dual therapy of pregnanolone and diazepam were not significantly different from control and similar to control were significantly different from diazepam monotherapy (*P < 0.05). GB/DZP, GB plus diazepam; GB/DZP/PREG, GB plus diazepam and pregnanolone; FT, fiber tracts; AMY, amygdala; THAL, thalamus; HIP, hippocampus; PIR, piriform cortex

Figure 7

NeuN cell density in rat brains collected 1 or 3 mo after whole‐body exposure to GB followed by atropine sulfate and HI‐6 at onset of toxic signs and diazepam with or without pregnanolone 30 min after seizure onset. Representative images of NeuN‐stained coronal brain sections in control rats not exposed to GB (Air control/NoGB; A‐C), rats exposed to GB and treated with delayed diazepam monotherapy (GB/DZP; D‐F), and rats exposed to GB and treated with delayed diazepam and pregnanolone dual therapy (GB/DZP/PREG; G‐I). J, Bar graphs of average (±SD) NeuN cell density 1 mo after GB exposure showing fewer neurons in the medial and lateral thalamus and the piriform cortex of GB‐exposed rats treated with delayed diazepam monotherapy compared with air control (+P < 0.05). Rats that received delayed dual therapy of diazepam and pregnanolone did not have significant loss of neurons in the thalamus or piriform cortex compared with air control and in the thalamus had significantly more neurons compared with diazepam monotherapy (*P < 0.05). K, Bar graphs of average (±SD) NeuN cell density 3 mo after GB exposure showing that rats treated with pregnanolone and diazepam dual therapy had less neuronal loss in the medial and lateral thalamus compared with those that received diazepam monotherapy. Both GB exposure groups had significantly loss of neurons in the amygdala and piriform cortex compared with air control at 3 mo after GB exposure (+P < 0.05)