Article Figures & Data
Figures
- Fig. 1.
Repeated administration of MC by the PO and intraperitoneal routes does not overtly impact body weight gain or long-term stability of the fully kindled seizure after acquisition of the fully kindled state. (A) Over 72% of mice that enrolled in the corneal-kindling protocol achieved the fully kindled state within 3 to 4 weeks. Sham-kindled mice did not present with a behavioral seizure during any of the stimulation sessions. N = 47 CKM and N = 35 sham. (B) The long-term stability of the fully kindled seizure was not adversely impacted by repeated intraperitoneal MC or SAL administration during the 6-week testing period. (C) Animals were randomized to treatment group after acquisition of the fully kindled state, as defined by five consecutive Racine stage 5 seizures. Repeated administration of MC or SAL by the intraperitoneal or PO route to sham- or corneal-kindled mice was not associated with any overt impacts on percentage change (C) or raw change (D) in body weight gain during the 6-week treatment period. CKM: i.p. MC, n = 14; i.p. SAL, n = 12; PO MC, n = 5; PO SAL, n = 3; sham-kindled mice: i.p. MC, n = 13; i.p. SAL, n = 12; PO MC, n = 5; PO SAL, n = 5.
- Fig. 2.
(A) Clinical assessments were performed by a board-certified laboratory animal veterinarian at four time points during the MC/SAL treatment period. Examination 1 occurred 1 week after kindling acquisition, prior to MC/SAL administration. Examination 2 occurred after 1 week of MC/SAL administration. Examination 3 occurred after 2.5 weeks of MC/SAL administration. Examination 4 occurred after 6 weeks of MC/SAL administration. Clinical scores range from 0 to 11 and were based on assessment of mouse appearance, posture, behavior, and body condition by a veterinarian blinded to treatment group and kindling status (mean ± S.E.M.). (B) A cohort of female C57Bl/6J mice (n = 10) were infected with influenza virus and assessed for clinical score on the same day as examination 2 of the sham PO SAL-treated mice. Acute infection of mice with influenza virus results in a significantly greater clinical score than sham-kindled CF-1 mice administered saline by the oral route (t = 25.0, P < 0.0001). ****Significantly greater than Sham PO SAL (P < 0.0001).
- Fig. 3.
Repeated administration of MC or SAL by the intraperitoneal route to sham- and corneal-kindled mice does not adversely affect exploratory behavior in an OF (mean ± S.E.M.). OF behavior was assessed by an automated program to quantify total distance traveled (A), percentage of distance traveled in the center of an OF (B), percentage of vertical rearing activity in the OF center (C), and percentage of total rest time in the center of the OF (D).
- Fig. 4.
Representative sections from kidney (A and B), liver (C and D), and carotid artery (E and F). (A, C, and E) Normal tissues from an intraperitoneal SAL-treated mouse. (A) Note that male mice have cuboidal parietal epithelium lining the glomerular capsule (asterisk). (B, D, and F) Tissues from an intraperitoneal MC-treated mouse; insets show high-magnification examples of FCs. (B) Glomerulus containing large foamy cells (arrow), enlarged with proliferative glomerulonephropathy. (D) Liver foci of FCs with surrounding mononuclear inflammatory cells. (Inset) High magnification of FCs without attending inflammation. (F) Carotid artery with abundant subintimal accumulations of FCs (arrow); lumen indicated (asterisk). (A–D) Original magnification, 200×. (E and F) Original magnification, 100×. (Insets) Original magnification, 400×.
- Fig. 5.
FCs are present in multiple organs and may be associated with inflammation and tissue damage. (A) Carotid artery. Note abundant intimal and subintimal accumulation of FCs, with FCs present within the lumen (asterisk). Original magnification, 400×. (B) Choroid plexus contains moderate perivascular accumulations of FCs (arrow). Original magnification, 100×. (C) Liver with coalescing microgranulomas. Original magnification, 200×. (D) Liver Mason’s trichrome stain for fibrosis (blue stain); there is a small amount of increased connective tissue (blue) adjacent to foci of FCs. Original magnification, 400×. (E) Mesentery. Mixed inflammatory foci adjacent to accumulations of FCs (arrow). Original magnification, 400×. (F) Glomerulonephritis (asterisk) and FCs within affected glomeruli (arrow). Original magnification, 400×. (Inset) PAS-stained glomerulus with intracapillary FCs. The cells are PAS negative. Original magnification, 400×.
- Fig. 6.
Administration of MC results in FC accumulation when administered by the intraperitoneal route, but not the PO route, regardless of kindling status. Liver, lung, kidney, spleen, mesentery, arteries, and choroid were assessed histologically by a veterinary pathologist masked to experimental treatment. A total disease score, ranging from 0 to 56, was developed to capture the presence and extent of FCs in the tissues as well as the presence or absence of degeneration and/or necrosis associated with the FC aggregates (mean ± S.E.M.).
Tables
Score 0 1 2 3 Appearance Normal Lack of grooming, sunken eyes, mild blepharitis/blepharospasm, mild hunched posture Ocular discharge, corneal ulceration, marked blepharitis/blepharospasm, prolonged hunched posture, abdominal distention, head tilt Hunched unmoving posture, labored breathing Clinical signs None Elevated or decreased respiratory and/or effort Clinical dehydration, marked pallor Behavior Normal Decreased interaction with cagemates, slow to move about cage, decreased interest in environment Isolated from cagemates, slow to move when stimulated, impaired mobility, ataxia, hypo- or hyperesthetic when handled Immobile, weakly or not responsive to handling, convulsing Body condition BCS ≥3 BCS 2 to 3 BCS 1 to 2 BCS ≤1 BCS, Body Condition Score.
FC Score Description 0 No FCs present 1 Scattered individual FCs present within the tissue (1–5 cells) 2 Clusters of FCs present, no inflammation 3 Clusters of FCs present with inflammation, or coalescing clusters of FCs without inflammation 4 Coalescing clusters of FCs with inflammation Tissue damage multiplier 1 No tissue damage present 2 Presence of degeneration or necrosis - TABLE 3
Complete blood count and white blood cell differential
Analytes were assessed across treatment groups by one-way ANOVA followed by Tukey’s post hoc analysis. Significant differences in pairwise comparisons among groups for individual analytes are demonstrated using superscript letters. Values are the mean ± S.D.
Test CKM i.p. MC (n = 6) CKM i.p. SAL (n = 5) Sham i.p. MC (n = 5) Sham i.p. SAL (n = 4) CKM PO MC (n = 3) Sham PO MC (n = 5) Sham PO SAL (n = 5) White blood cells (K/μl) 7.7 ± 1.4 7.6 ± 2.9 7.8 ± 1.4 6.8 ± 2.6 4.7 ± 1.7 4.7 ± 0.9 5.0 ± 1.4 Red blood cells (M/μl) 9.1 ± 0.6 9.0 ± 0.4 8.6 ± 0.4 8.9 ± 1.2 9.4 ± 0.5 8.7 ± 1.5 8.6 ± 1.6 Hemoglobin (g/dl) 13.8 ± 0.8 12.7 ± 3.4 13.1 ± 0.7 13.7 ± 2.0 15.5 ± 0.7 14.6 ± 2.8 14.3 ± 2.5 Hematocrit (%) 56.0 ± 4.5 55.0 ± 3.1 52.0 ± 2.8 54.3 ± 7.1 60.1 ± 4.1 52.6 ± 8.8 53.7 ± 10.1 Mean corpuscular volume (fl) 61.6 ± 2.0 60.8 ± 2.4 60.4 ± 2.3 60.8 ± 1.0 64.1 ± 2.6 60.6 ± 1.9 62.7 ± 2.1 Mean corpuscular hemoglobin (pg) 15.2 ± 0.4a,b,c 15.5 ± 0.5d,e,f 15.3 ± 0.5g,h,i 15.3 ± 0.4j,k,l 16.5 ± 0.3c,f,i,l 16.5 ± 0.4a,d,g,j 16.7 ± 0.3b,e,h,k Mean corpuscular hemoglobin concentration (%) 24.8 ± 1.1a,b 25.5 ± 0.7d 25.2 ± 0.8g 25.2 ± 0.5k 25.8 ± 0.8 27.3 ± 0.4a,d,g,k 26.7 ± 0.7b Neutrophils % 33.3 ± 7.1a,b,c 40.8 ± 6.7d,e,f 29.2 ± 3.8g,h,i 27.8 ± 14.4j,k 12.3 ± 4.0c,f,i 10.0 ± 1.6a,d,g,j 10.8 ± 1.3b,e,h,k #/μl 2597.5 ± 801.3a,b,c 3095.6 ± 1281.3d,e,f 2270.2 ± 521.9g,h 1858.5 ± 1056.3 540.3 ± 174.0c,f 462.4 ± 96.5a,d,g 533.8 ± 145.6b,e,h Lymphocytes % 59.7 ± 9.2a,b,c 55.0 ± 9.1d,e,f 62.0 ± 8.5g,h,i 67.5 ± 18.6j,k 85.3 ± 4.0c,f,i 87.2 ± 0.8a,d,g,j 87.2 ± 2.4b,e,h,k #/μl 4595.8 ± 974.3 4164.8 ± 1708.8 4816.2 ± 1038.9 4556.3 ± 2305.3 4004.0 ± 1534.6 4065.0 ± 807.1 4312.2 ± 1182.3 Monocytes % 6.8 ± 3.1 3.6 ± 2.7 8.8 ± 5.0g,h 4.0 ± 4.2 2.0 ± 1.7 1.2 ± 0.8g 1.6 ± 1.9h #/μl 526.3 ± 224.7a 248.4 ± 155.4 693.6 ± 452.6g,h,i 294.8 ± 299.7 103.7 ± 108.6i 55.0 ± 43.5a,g 91.2 ± 116.5h Eosinophils % 0.2 ± 0.4a 0.4 ± 0.5 0.0 ± 0.0g 0.8 ± 1.0 0.3 ± 0.6 1.6 ± 0.9a,g 0.4 ± 0.5 #/μl 13.7 ± 33.5 31.2 ± 45.5 0.0 ± 0.0 40.5 ± 48.3 18.7 ± 32.3 77.6 ± 54.1 22.8 ± 31.6 Basophils % 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 #/μl 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Platelets (K/μl) 921.3 ± 411.0 652.4 ± 430.2 1067.6 ± 389.0 813.5 ± 398.9 1063.7 ± 382.8 534.2 ± 407.6 485.6 ± 374.3 ↵a CKM MC i.p. vs. sham MC PO.
↵b CKM MC i.p. vs. sham SAL PO.
↵c CKM MC i.p. vs. CKM MC PO.
↵d CKM SAL i.p. vs. sham MC PO.
↵e CKM SAL i.p. vs. sham SAL PO.
↵f CKM SAL i.p. vs. CKM MC PO.
↵g Sham MC i.p. vs. sham MC PO.
↵h Sham MC i.p. vs. sham SAL PO.
↵i Sham MC i.p. vs. CKM MC PO.
↵j Sham SAL i.p. vs. sham MC PO.
↵k Sham SAL i.p. vs. sham SAL PO.
↵l Sham SAL i.p. vs. CKM MC PO.
- TABLE 4
Serum biochemistry analytes
Analytes were assessed across treatment groups by one-way ANOVA followed by Tukey’s post hoc analysis. Significant differences in pairwise comparisons among groups for individual analytes are demonstrated using superscript letters. Values are the mean ± S.D.
Test CKM i.p. MC (n = 6) CKM i.p. SAL (n = 5) Sham i.p. MC (n = 5) Sham i.p. SAL (n = 4) CKM PO MC (n = 3) Sham PO MC (n = 5) Sham PO SAL (n = 5) Glucose (mg/dl) 274.5 ± 44.5 282.0 ± 49.6 319.2 ± 108.2 268.0 ± 53.5 281.0 ± 48.7 357.0 ± 77.7 269.6 ± 55.5 Blood urea nitrogen (mg/dl) 25.2 ± 1.8a 21.6 ± 3.3 26.0 ± 3.5b 22.5 ± 3.3 22.7 ± 3.8 19.4 ± 2.1a,b 22.2 ± 2.6 Calcium (mg/dl) 10.8 ± 0.9 10.9 ± 0.6 11.4 ± 0.5 10.7 ± 0.3 10.3 ± 0.4 11.3 ± 0.7 10.4 ± 0.4 Phosphate (mg/dl) 14.7 ± 1.9 14.3 ± 1.4 13.9 ± 1.1 14.2 ± 0.3 12.8 ± 2.2 14.6 ± 1.8 13.3 ± 0.7 Total protein (g/dl) 6.1 ± 0.5 6.1 ± 0.5 5.8 ± 0.4 5.9 ± 0.3 5.7 ± 0.2 5.8 ± 0.2 5.5 ± 0.3 Albumin (g/dl) 3.5 ± 0.2 3.5 ± 0.2 3.2 ± 0.2 3.3 ± 0.1 3.5 ± 0.3 3.5 ± 0.2 3.5 ± 0.2 Globulin (g/dl) 2.6 ± 0.3c 2.6 ± 0.3 2.6 ± 0.2d 2.6 ± 0.3e 2.2 ± 0.1 2.3 ± 0.1 2.0 ± 0.2c,d,e Total bilirubin (mg/dl) 0.2 ± 0.1 0.2 ± 0.1 0.1 ± 0.0 0.2 ± 0.1 0.2 ± 0.1 0.2 ± 0.1 0.1 ± 0.1 Alkaline phosphatase (U/l) 125.5 ± 43.3 178.4 ± 57.9f 83.6 ± 26.4f,b 157.0 ± 41.0 132.0 ± 25.2 189.8 ± 65.4b 152.6 ± 24.5 Alanine aminotransferase (U/l) 54.7 ± 25.8 55.6 ± 24.4 43.8 ± 10.1 40.8 ± 11.8 46.0 ± 12.8 37.6 ± 13.1 37.0 ± 7.3 Aspartate aminotransferase (U/l) 251.2 ± 335.9 100.4 ± 36.7 62.6 ± 19.8 85.0 ± 31.8 70.0 ± 14.0 70.2 ± 40.4 89.4 ± 66.2 Cholesterol (mg/dl) 142.2 ± 40.8 148.0 ± 26.1 142.0 ± 18.5 149.0 ± 17.4 133.3 ± 16.0 165.8 ± 18.5 138.2 ± 27.3
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