Micro image

The gray-matter area was determined by measuring the MAP2 immunoreactive area from eight serial sections per animal. Brain injury was evaluated by the volume of infarction and total hemispheric tissue loss and neuropathological scoring, which was performed by a person who did not have prior knowledge of the groups. The MAP2-positive and negative areas in each section were measured in both hemispheres using Micro Image (Olympus, Japan). The tissue volume was calculated from the MAP2-positive or negative areas according to the Cavalieri principle using the following formula: V = ΣA × P × T, where V = the total volume, ΣA = the sum of area measurements, P = the inverse of the sampling fraction, and T = the section thickness. The total hemispheric tissue loss was calculated as the MAP2-positive volume in the contralateral hemisphere minus the MAP2-positive volume in the ipsilateral hemisphere. The neuropathological score of gray matter from different brain regions was assessed. Briefly, the cortical injury was graded from 0 to 4 with 0 being no observable injury and 4 indicating confluent infarction. The injury in the hippocampus, striatum, and thalamus was assessed both with respect to hypotrophy (scored from 0 to 3) and injury/infarction (scored from 0 to 3), resulting in a total possible score of 22^{24 (link)}.

Brain injury was evaluated using the total tissue loss volume and neuropathological scoring by a person who did not have prior knowledge of the groups. The MAP2-positive area in each section was measured in both hemispheres using Micro Image (Olympus, Japan). The volume was calculated from the MAP2-positive areas according to the Cavalieri principle, using the formula: V =  SA*T*P, where V is the total volume, SA is the sum of area measurements, T is the section thickness and P is the inverse of the sampling fraction. The total tissue loss was defined as the MAP2-positive volume in the contralateral hemisphere minus the MAP2-positive volume in the ipsilateral hemisphere. The neuropathological score for different brain regions was assessed. Briefly, the cortical injury was graded from 0 to 4 with 0 being no observable injury and 4 indicating confluent infarction. The injury in the hippocampus, striatum, and thalamus was assessed both with respect to hypotrophy (scored from 0 to 3) and injury/infarction (scored from 0 to 3), resulting in a total score of 22.

Area contours were drawn and measured in every 50^th section. The active caspase-3 and AIF-positive cells (DCA = 7, vehicle = 8, 24 h) were counted at 400× magnification in the border zone of the injured cortex within an area of 0.196 mm² (one visual field) using Micro Image (Olympus, Japan). Three sections were counted from each brain with an interval of 250 μm. The average was defined as n = 1 when comparing different brains. All of the counting was carried out by investigators blinded to group assignment.

Specimens were examined with an Olympus SZ61 stereomicroscope, measurements were taken with an ocular grid. The habitus photographs were taken using a Canon MP-E 65mm/2.8 1–5× Macro lens attached to a Canon EOS 550D camera. Partially focused images of each specimen were combined using Zerene stacker software. Male genitalia images were taken with a Provis AX70 (Olympus) microscope with digital image processing capability using Micro Image (Olympus) software.
Specimens of the newly described species are provided with one printed red label:
“Glaresis | hespericula sp. nov. | HOLOTYPUS ♂ [or] PARATYPUS ♀ | David Král & Lucie Hrůzová 2018”. Both type specimens are deposited in the National Museum Praha, Czech Republic.
Exact label data are cited for the type material examined. Lines within each label are separated by a single vertical bar “|”. Information in quotation marks indicates the original spelling. Our remarks and additional comments are placed in brackets.
For morphological terms used in the description we largely follow Gordon and Hanley (2014) and Král et al. (2017) (link).

Area contours were drawn and measured in every 50th section. The active caspase-3-positive and AIF-positive nuclei and the 8-OHG-positive cells were counted at ×400 magnification in the border zone of the injured cortex and striatum, cornu ammonis area 1 (CA1), and NH within an area of 0.196 mm² (one visual field) using Micro Image (Olympus, Japan). Three sections were counted from each brain with an interval of 250 μm^{39 (link)}. The numbers of Ki-67-positive and DCX-positive cells were counted in the SGZ of the dentate gyrus in three adjacent sections in the hippocampus. The length of the SGZ was measured, and the number of positive cells was expressed as the number per mm. The average was defined as n = 1 when comparing different brains. All of the counting was carried out by investigators blinded to group assignment.

Brain injury was evaluated based on MAP2 and MBP immunostainings. Both hemispheres of each section were measured using Micro Image (Olympus, Japan). MAP2-positive and negative and MBP-positive tissue volume calculations as well as neuropathological scores of the gray matter from different brain regions were assessed as described previously^{11 (link)}. The infarction volume was equal to the MAP2-negative volume in the IL hemisphere. MBP tissue loss ratio was calculated as: ((CL hemisphere − IL hemisphere)/CL hemisphere) × 100%. All evaluations were carried out by investigators blinded to group assignment.