39c1ad849b7cf045b1ca419fbbb657cf pmc_10604167.pdf 0dda89e9dd29dc1220ecaf15ac2f032f7434fdd9 pmc_10604167.pdf a407dc647fed15a30de137574faf3167d8d22eabdde8ab8d46a4183bcc0a297c pmc_10604167.pdf Title: Genetic and Pharmacological Blockade of Sigma-1 Receptors Attenuates Inflammation-Associated Hypersensitivity during Acute Colitis in CD1 Mice Subject: Sigma-1 receptors (1Rs) are implicated in nociception, including pain sensitization, and inflammation. We assessed the role of 1Rs on acute colitis-associated hypersensitivity using both genetic (constitutive knockout) and pharmacological blockade of the receptor. Colitis was induced in CD1 wild-type (WT) and 1R KO mice (exposure to dextran sodium sulfate, 3%). A von Frey test was used to assess referred mechanosensitivity (abdominal and plantar withdrawal responses). The effects of the selective 1R antagonists BD1063 and E-52862 were also assessed in WT animals. The expression of immune and sensory-related markers (RT-qPCR, Western blot) was assessed in the colon and lumbosacral spinal cord. The genetic ablation or pharmacological blockade of 1Rs attenuated acute colonic inflammation in a similar manner. Mechanosensitivity was similar in WT and 1R KO mice before colitis. In WT mice, but not in 1R KO, colitis was associated with the development of referred mechanical hypersensitivity, manifested as a reduction in the withdrawal thresholds to mechanical probing (paw and abdominal wall). In WT mice, BD1063 and E-52862 blocked colitis-associated hypersensitivity. A genotype- and treatment-related differential regulation of sensory-related markers was detected locally (colon) and within the spinal cord. 1Rs are involved in the development of acute intestinal inflammation and its associated referred mechanical hypersensitivity. The selective modulation of sensory-related pathways within the colon and spinal cord might be part of the underlying mechanisms. These observations support the pharmacological use of 1R antagonists for the treatment of intestinal inflammation-induced hypersensitivity. Keywords: BD1063; colitis; E-52862; hypersensitivity; intestinal inflammation; pain; sigma 1 receptor; visceral pain Author: Sergio López-Estévez, Mònica Aguilera, Georgia Gris, Beatriz de la Puente, Alicia Carceller and Vicente Martínez Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Oct 26 03:19:03 2023 CEST ModDate: Thu Oct 26 03:21:02 2023 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 22 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 4740570 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- CMFVHJ+VnURWPalladioL Type 1 Custom yes yes no 10 0 UVKXNJ+EURM10 Type 1 Builtin yes yes no 15 0 SEMBIN+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 FSAHCE+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 YXOEBV+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 RNXXBO+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 EBFBTV+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 50 0 WTAUCZ+CMSY10 Type 1 Builtin yes yes no 72 0 CIDFont+F3 CID TrueType Identity-H yes no yes 81 0 CIDFont+F1 CID TrueType Identity-H yes no yes 89 0 CIDFont+F2 CID TrueType Identity-H yes no yes 97 0 KSGHMQ+PazoMath Type 1 Builtin yes yes no 113 0 SUZJEW+PazoMath-Italic Type 1 Builtin yes yes no 120 0 CIDFont+F3 CID TrueType Identity-H yes no yes 129 0 CIDFont+F1 CID TrueType Identity-H yes no yes 137 0 CIDFont+F2 CID TrueType Identity-H yes no yes 145 0 CIDFont+F4 CID TrueType Identity-H yes no yes 153 0 CIDFont+F3 CID TrueType Identity-H yes no yes 338 0 CIDFont+F1 CID TrueType Identity-H yes no yes 346 0 CIDFont+F2 CID TrueType Identity-H yes no yes 354 0 CIDFont+F3 CID TrueType Identity-H yes no yes 463 0 CIDFont+F1 CID TrueType Identity-H yes no yes 471 0 CIDFont+F2 CID TrueType Identity-H yes no yes 479 0 CIDFont+F3 CID TrueType Identity-H yes no yes 542 0 CIDFont+F1 CID TrueType Identity-H yes no yes 550 0 CIDFont+F2 CID TrueType Identity-H yes no yes 558 0 CIDFont+F5 CID TrueType Identity-H yes no yes 566 0 CIDFont+F3 CID TrueType Identity-H yes no yes 631 0 CIDFont+F1 CID TrueType Identity-H yes no yes 639 0 CIDFont+F2 CID TrueType Identity-H yes no yes 647 0 CIDFont+F3 CID TrueType Identity-H yes no yes 727 0 CIDFont+F1 CID TrueType Identity-H yes no yes 735 0 CIDFont+F2 CID TrueType Identity-H yes no yes 743 0 CIDFont+F4 CID TrueType Identity-H yes no yes 751 0 CIDFont+F3 CID TrueType Identity-H yes no yes 922 0 CIDFont+F1 CID TrueType Identity-H yes no yes 930 0 CIDFont+F2 CID TrueType Identity-H yes no yes 938 0 CIDFont+F3 CID TrueType Identity-H yes no yes 1081 0 CIDFont+F1 CID TrueType Identity-H yes no yes 1089 0 CIDFont+F2 CID TrueType Identity-H yes no yes 1097 0 CIDFont+F3 CID TrueType Identity-H yes no yes 1242 0 CIDFont+F1 CID TrueType Identity-H yes no yes 1250 0 CIDFont+F2 CID TrueType Identity-H yes no yes 1258 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-11-18 02:42:13 CET RepresentationInformation: pmc_10604167.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-11-08 02:28:14 CET Size: 4740570 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 1677 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Children: Item: Title: Animals Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Colitis Induction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Drugs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Evaluation of Referred Mechanical Hypersensitivity: Von Frey Test Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Experimental Protocols Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: Sample Collection Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: Clinical and Macroscopic Assessment of Inflammation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: Histological Studies Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Item: Title: Gene Expression: Quantitative Reverse Transcription-PCR Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@e720b71 Item: Title: Protein Expression: Western Blot Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1b26f7b2 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@491cc5c9 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@74ad1f1f Children: Item: Title: 1R KO CD1 Mice Develop an Attenuated Acute Colitis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6a1aab78 Item: Title: 1R KO Mice Do Not Develop Acute Colitis-Associated Hypersensitivity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@462d5aee Item: Title: Antagonism of 1Rs with BD1063 or E-52862 Attenuates DSS-Induced Colitis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@69b0fd6f Item: Title: Antagonism of 1Rs with BD1063 or E-52862 Attenuated Colitis-Associated Hypersensitivity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@757942a1 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4a87761d Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@66d1af89 Info: Title: Genetic and Pharmacological Blockade of Sigma-1 Receptors Attenuates Inflammation-Associated Hypersensitivity during Acute Colitis in CD1 Mice Author: Sergio López-Estévez, Mònica Aguilera, Georgia Gris, Beatriz de la Puente, Alicia Carceller and Vicente Martínez Subject: Sigma-1 receptors (1Rs) are implicated in nociception, including pain sensitization, and inflammation. We assessed the role of 1Rs on acute colitis-associated hypersensitivity using both genetic (constitutive knockout) and pharmacological blockade of the receptor. Colitis was induced in CD1 wild-type (WT) and 1R KO mice (exposure to dextran sodium sulfate, 3%). A von Frey test was used to assess referred mechanosensitivity (abdominal and plantar withdrawal responses). The effects of the selective 1R antagonists BD1063 and E-52862 were also assessed in WT animals. The expression of immune and sensory-related markers (RT-qPCR, Western blot) was assessed in the colon and lumbosacral spinal cord. The genetic ablation or pharmacological blockade of 1Rs attenuated acute colonic inflammation in a similar manner. Mechanosensitivity was similar in WT and 1R KO mice before colitis. In WT mice, but not in 1R KO, colitis was associated with the development of referred mechanical hypersensitivity, manifested as a reduction in the withdrawal thresholds to mechanical probing (paw and abdominal wall). In WT mice, BD1063 and E-52862 blocked colitis-associated hypersensitivity. A genotype- and treatment-related differential regulation of sensory-related markers was detected locally (colon) and within the spinal cord. 1Rs are involved in the development of acute intestinal inflammation and its associated referred mechanical hypersensitivity. The selective modulation of sensory-related pathways within the colon and spinal cord might be part of the underlying mechanisms. These observations support the pharmacological use of 1R antagonists for the treatment of intestinal inflammation-induced hypersensitivity. Keywords: BD1063; colitis; E-52862; hypersensitivity; intestinal inflammation; pain; sigma 1 receptor; visceral pain Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Oct 26 03:19:03 CEST 2023 ModDate: Thu Oct 26 03:21:02 CEST 2023 ID: 0x4cada0d2c2befa9a889aa105c7ca7ad8, 0x4cada0d2c2befa9a889aa105c7ca7ad8 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: RNXXBO+VnURWPalladioL-Bold FontSubset: true FirstChar: 178 LastChar: 226 FontDescriptor: FontName: RNXXBO+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: KSGHMQ+PazoMath FontSubset: true FirstChar: 68 LastChar: 83 FontDescriptor: FontName: KSGHMQ+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true Font: BaseFont: SEMBIN+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: SEMBIN+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: FSAHCE+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 246 FontDescriptor: FontName: FSAHCE+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: WTAUCZ+CMSY10 FontSubset: true FirstChar: 0 LastChar: 14 FontDescriptor: FontName: WTAUCZ+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: SUZJEW+PazoMath-Italic FontSubset: true FirstChar: 115 LastChar: 115 FontDescriptor: FontName: SUZJEW+PazoMath-Italic Flags: Symbolic FontBBox: -70, -277, 902, 733 FontFile: true Font: BaseFont: CMFVHJ+VnURWPalladioL FontSubset: true FirstChar: 129 LastChar: 226 FontDescriptor: FontName: CMFVHJ+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: YXOEBV+URWPalladioL-Ital FontSubset: true FirstChar: 3 LastChar: 151 FontDescriptor: FontName: YXOEBV+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: UVKXNJ+EURM10 FontSubset: true FirstChar: 12 LastChar: 34 FontDescriptor: FontName: UVKXNJ+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 54, 279, 152, 288 Annotation: Subtype: Link Rect: 34, 269, 105, 276 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 126, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 147, 87 Annotation: Subtype: Link Rect: 34, 756, 197, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 240, 644, 251, 657 Annotation: Subtype: Link Rect: 139, 633, 150, 645 Annotation: Subtype: Link Rect: 253, 163, 260, 172 ActionDest: 1384 Annotation: Subtype: Link Rect: 263, 163, 270, 172 ActionDest: 1384 Annotation: Subtype: Link Rect: 426, 126, 433, 135 ActionDest: 1384 Annotation: Subtype: Link Rect: 434, 126, 441, 135 ActionDest: 1384 Annotation: Subtype: Link Rect: 190, 88, 197, 97 ActionDest: 1384 Annotation: Subtype: Link Rect: 197, 88, 204, 97 ActionDest: 1384 Annotation: Subtype: Link Rect: 130, 17, 302, 28 Annotation: Subtype: Link Rect: 389, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 388, 87, 407 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 222, 698, 229, 707 ActionDest: 1384 Annotation: Subtype: Link Rect: 229, 698, 236, 707 ActionDest: 1384 Annotation: Subtype: Link Rect: 237, 698, 244, 707 ActionDest: 1384 Annotation: Subtype: Link Rect: 310, 673, 317, 682 ActionDest: 1384 Annotation: Subtype: Link Rect: 446, 648, 453, 657 ActionDest: 1384 Annotation: Subtype: Link Rect: 456, 647, 463, 657 ActionDest: 1384 Annotation: Subtype: Link Rect: 329, 597, 336, 606 ActionDest: 1384 Annotation: Subtype: Link Rect: 339, 597, 346, 606 ActionDest: 1384 Annotation: Subtype: Link Rect: 368, 572, 380, 581 ActionDest: 1384 Annotation: Subtype: Link Rect: 380, 572, 392, 581 ActionDest: 1384 Annotation: Subtype: Link Rect: 387, 522, 399, 531 ActionDest: 1384 Annotation: Subtype: Link Rect: 399, 522, 411, 531 ActionDest: 1384 Annotation: Subtype: Link Rect: 411, 522, 423, 531 ActionDest: 1384 Annotation: Subtype: Link Rect: 396, 497, 403, 506 ActionDest: 1384 Annotation: Subtype: Link Rect: 403, 497, 415, 506 ActionDest: 1384 Annotation: Subtype: Link Rect: 418, 497, 430, 506 ActionDest: 1384 Annotation: Subtype: Link Rect: 187, 434, 199, 443 ActionDest: 1384 Annotation: Subtype: Link Rect: 199, 434, 211, 443 ActionDest: 1384 Annotation: Subtype: Link Rect: 544, 422, 556, 431 ActionDest: 1384 Annotation: Subtype: Link Rect: 336, 245, 348, 254 ActionDest: 1384 Annotation: Subtype: Link Rect: 211, 207, 222, 216 ActionDest: 1384 Annotation: Subtype: Link Rect: 520, 195, 532, 203 ActionDest: 1384 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 412, 670, 424, 679 ActionDest: 1384 Annotation: Subtype: Link Rect: 424, 670, 436, 679 ActionDest: 1384 Annotation: Subtype: Link Rect: 231, 607, 242, 616 ActionDest: 1384 Annotation: Subtype: Link Rect: 334, 594, 345, 603 ActionDest: 1387 Annotation: Subtype: Link Rect: 466, 556, 478, 565 ActionDest: 1387 Annotation: Subtype: Link Rect: 478, 557, 489, 565 ActionDest: 1387 Annotation: Subtype: Link Rect: 308, 468, 320, 477 ActionDest: 1384 Annotation: Subtype: Link Rect: 242, 380, 254, 389 ActionDest: 1384 Annotation: Subtype: Link Rect: 255, 380, 267, 389 ActionDest: 1384 Annotation: Subtype: Link Rect: 267, 380, 279, 389 ActionDest: 1387 Annotation: Subtype: Link Rect: 531, 368, 543, 377 ActionDest: 1384 Annotation: Subtype: Link Rect: 544, 368, 556, 377 ActionDest: 1384 Annotation: Subtype: Link Rect: 484, 343, 496, 352 ActionDest: 1387 Annotation: Subtype: Link Rect: 497, 343, 509, 352 ActionDest: 1387 Annotation: Subtype: Link Rect: 329, 315, 336, 327 ActionDest: 76 Annotation: Subtype: Link Rect: 437, 85, 443, 99 ActionDest: 107 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 276, 451, 283, 463 ActionDest: 107 Annotation: Subtype: Link Rect: 251, 426, 258, 438 ActionDest: 76 Annotation: Subtype: Link Rect: 459, 429, 471, 438 ActionDest: 1384 Annotation: Subtype: Link Rect: 471, 429, 483, 438 ActionDest: 1387 Annotation: Subtype: Link Rect: 484, 429, 496, 438 ActionDest: 1387 Annotation: Subtype: Link Rect: 547, 363, 554, 375 ActionDest: 76 Annotation: Subtype: Link Rect: 395, 338, 402, 350 ActionDest: 107 Annotation: Subtype: Link Rect: 549, 275, 556, 287 ActionDest: 107 Annotation: Subtype: Link Rect: 464, 225, 471, 237 ActionDest: 76 Annotation: Subtype: Link Rect: 502, 225, 509, 237 ActionDest: 107 Annotation: Subtype: Link Rect: 290, 125, 297, 137 ActionDest: 76 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 399, 597, 411, 606 ActionDest: 1384 Annotation: Subtype: Link Rect: 425, 522, 437, 531 ActionDest: 1384 Annotation: Subtype: Link Rect: 457, 421, 468, 430 ActionDest: 1384 Annotation: Subtype: Link Rect: 469, 421, 481, 430 ActionDest: 1387 Annotation: Subtype: Link Rect: 420, 132, 432, 141 ActionDest: 1387 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 230, 557, 237, 569 ActionDest: 117 Annotation: Subtype: Link Rect: 369, 519, 376, 531 ActionDest: 117 Annotation: Subtype: Link Rect: 357, 95, 364, 107 ActionDest: 124 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 338, 101, 345, 113 ActionDest: 124 Annotation: Subtype: Link Rect: 315, 76, 322, 88 ActionDest: 124 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 292, 695, 299, 707 ActionDest: 124 Annotation: Subtype: Link Rect: 399, 645, 406, 657 ActionDest: 124 Annotation: Subtype: Link Rect: 420, 570, 427, 582 ActionDest: 333 Annotation: Subtype: Link Rect: 196, 532, 202, 544 ActionDest: 333 Annotation: Subtype: Link Rect: 520, 507, 527, 519 ActionDest: 333 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 469, 670, 476, 682 ActionDest: 458 Annotation: Subtype: Link Rect: 383, 68, 390, 80 ActionDest: 458 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 197, 645, 204, 657 ActionDest: 537 Annotation: Subtype: Link Rect: 196, 607, 203, 619 ActionDest: 537 Annotation: Subtype: Link Rect: 242, 557, 249, 569 ActionDest: 537 Annotation: Subtype: Link Rect: 530, 85, 537, 97 ActionDest: 626 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 345, 733, 352, 745 ActionDest: 626 Annotation: Subtype: Link Rect: 224, 239, 231, 251 ActionDest: 722 Annotation: Subtype: Link Rect: 456, 202, 463, 214 ActionDest: 722 Annotation: Subtype: Link Rect: 489, 126, 496, 138 ActionDest: 722 Annotation: Subtype: Link Rect: 392, 76, 399, 88 ActionDest: 722 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 549, 695, 556, 707 ActionDest: 917 Annotation: Subtype: Link Rect: 324, 632, 331, 644 ActionDest: 917 Annotation: Subtype: Link Rect: 318, 595, 325, 607 ActionDest: 917 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 488, 235, 495, 247 ActionDest: 1076 Annotation: Subtype: Link Rect: 199, 173, 206, 185 ActionDest: 1076 Annotation: Subtype: Link Rect: 459, 135, 466, 147 ActionDest: 1076 Page: Sequence: 14 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 352, 683, 364, 695 ActionDest: 1237 Annotation: Subtype: Link Rect: 433, 657, 445, 669 ActionDest: 1237 Annotation: Subtype: Link Rect: 496, 595, 508, 607 ActionDest: 1237 Annotation: Subtype: Link Rect: 513, 570, 525, 582 ActionDest: 1237 Annotation: Subtype: Link Rect: 199, 494, 210, 506 ActionDest: 1237 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 183, 707, 195, 716 ActionDest: 1384 Annotation: Subtype: Link Rect: 195, 707, 207, 716 ActionDest: 1387 Annotation: Subtype: Link Rect: 208, 707, 220, 716 ActionDest: 1387 Annotation: Subtype: Link Rect: 484, 569, 491, 578 ActionDest: 1384 Annotation: Subtype: Link Rect: 494, 569, 501, 578 ActionDest: 1384 Annotation: Subtype: Link Rect: 202, 507, 214, 516 ActionDest: 1384 Annotation: Subtype: Link Rect: 215, 506, 227, 516 ActionDest: 1384 Annotation: Subtype: Link Rect: 476, 494, 488, 503 ActionDest: 1387 Annotation: Subtype: Link Rect: 489, 494, 501, 503 ActionDest: 1387 Annotation: Subtype: Link Rect: 217, 431, 229, 440 ActionDest: 1387 Annotation: Subtype: Link Rect: 229, 431, 241, 440 ActionDest: 1387 Annotation: Subtype: Link Rect: 379, 406, 391, 415 ActionDest: 1384 Annotation: Subtype: Link Rect: 259, 293, 271, 302 ActionDest: 1384 Annotation: Subtype: Link Rect: 405, 280, 417, 290 ActionDest: 1384 Annotation: Subtype: Link Rect: 205, 255, 217, 264 ActionDest: 1387 Annotation: Subtype: Link Rect: 218, 255, 230, 264 ActionDest: 1387 Annotation: Subtype: Link Rect: 251, 230, 263, 239 ActionDest: 1387 Annotation: Subtype: Link Rect: 264, 230, 276, 239 ActionDest: 1387 Annotation: Subtype: Link Rect: 302, 143, 314, 151 ActionDest: 1384 Annotation: Subtype: Link Rect: 315, 143, 327, 151 ActionDest: 1384 Annotation: Subtype: Link Rect: 327, 142, 339, 151 ActionDest: 1384 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 289, 660, 301, 669 ActionDest: 1387 Annotation: Subtype: Link Rect: 484, 622, 496, 631 ActionDest: 1387 Annotation: Subtype: Link Rect: 519, 472, 531, 481 ActionDest: 1384 Annotation: Subtype: Link Rect: 531, 472, 543, 481 ActionDest: 1384 Annotation: Subtype: Link Rect: 544, 472, 556, 481 ActionDest: 1384 Annotation: Subtype: Link Rect: 495, 409, 507, 418 ActionDest: 1384 Annotation: Subtype: Link Rect: 508, 409, 520, 418 ActionDest: 1384 Annotation: Subtype: Link Rect: 520, 409, 532, 418 ActionDest: 1384 Annotation: Subtype: Link Rect: 367, 309, 374, 318 ActionDest: 1384 Annotation: Subtype: Link Rect: 375, 309, 382, 318 ActionDest: 1384 Annotation: Subtype: Link Rect: 382, 309, 389, 318 ActionDest: 1384 Annotation: Subtype: Link Rect: 390, 309, 402, 318 ActionDest: 1387 Annotation: Subtype: Link Rect: 544, 284, 556, 293 ActionDest: 1387 Annotation: Subtype: Link Rect: 190, 171, 202, 180 ActionDest: 1384 Annotation: Subtype: Link Rect: 202, 170, 214, 180 ActionDest: 1384 Annotation: Subtype: Link Rect: 252, 146, 264, 154 ActionDest: 1384 Annotation: Subtype: Link Rect: 264, 146, 276, 154 ActionDest: 1384 Annotation: Subtype: Link Rect: 277, 145, 289, 154 ActionDest: 1384 Annotation: Subtype: Link Rect: 289, 145, 301, 154 ActionDest: 1387 Annotation: Subtype: Link Rect: 302, 145, 314, 154 ActionDest: 1387 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 279, 735, 291, 744 ActionDest: 1387 Annotation: Subtype: Link Rect: 439, 723, 451, 732 ActionDest: 1387 Annotation: Subtype: Link Rect: 353, 685, 365, 694 ActionDest: 1387 Annotation: Subtype: Link Rect: 366, 685, 377, 694 ActionDest: 1387 Annotation: Subtype: Link Rect: 435, 635, 447, 644 ActionDest: 1387 Annotation: Subtype: Link Rect: 448, 635, 460, 644 ActionDest: 1387 Annotation: Subtype: Link Rect: 374, 585, 386, 594 ActionDest: 1384 Annotation: Subtype: Link Rect: 387, 585, 399, 594 ActionDest: 1384 Annotation: Subtype: Link Rect: 399, 585, 411, 594 ActionDest: 1384 Annotation: Subtype: Link Rect: 226, 509, 238, 519 ActionDest: 1387 Annotation: Subtype: Link Rect: 210, 434, 221, 443 ActionDest: 1391 Annotation: Subtype: Link Rect: 222, 434, 234, 443 ActionDest: 1391 Annotation: Subtype: Link Rect: 215, 346, 227, 355 ActionDest: 1391 Annotation: Subtype: Link Rect: 230, 346, 242, 355 ActionDest: 1391 Annotation: Subtype: Link Rect: 451, 233, 463, 242 ActionDest: 1391 Annotation: Subtype: Link Rect: 463, 233, 475, 242 ActionDest: 1391 Annotation: Subtype: Link Rect: 380, 208, 391, 217 ActionDest: 1387 Page: Sequence: 19 Annotations: Annotation: Subtype: Link Rect: 203, 698, 214, 707 ActionDest: 1384 Annotation: Subtype: Link Rect: 215, 698, 227, 707 ActionDest: 1391 Annotation: Subtype: Link Rect: 386, 685, 398, 694 ActionDest: 1391 Annotation: Subtype: Link Rect: 399, 685, 411, 694 ActionDest: 1391 Annotation: Subtype: Link Rect: 393, 647, 404, 657 ActionDest: 1384 Annotation: Subtype: Link Rect: 525, 483, 560, 494 Annotation: Subtype: Link Rect: 165, 471, 399, 482 Page: Sequence: 20 Annotations: Annotation: Subtype: Link Rect: 222, 707, 260, 718 Annotation: Subtype: Link Rect: 443, 684, 480, 695 Annotation: Subtype: Link Rect: 486, 684, 522, 695 Annotation: Subtype: Link Rect: 68, 662, 105, 672 Annotation: Subtype: Link Rect: 111, 662, 148, 672 Annotation: Subtype: Link Rect: 95, 639, 133, 649 Annotation: Subtype: Link Rect: 456, 626, 493, 638 Annotation: Subtype: Link Rect: 499, 626, 535, 638 Annotation: Subtype: Link Rect: 419, 615, 457, 626 Annotation: Subtype: Link Rect: 131, 581, 168, 591 Annotation: Subtype: Link Rect: 59, 558, 96, 568 Annotation: Subtype: Link Rect: 59, 535, 96, 545 Annotation: Subtype: Link Rect: 210, 500, 247, 511 Annotation: Subtype: Link Rect: 59, 466, 96, 476 Annotation: Subtype: Link Rect: 102, 466, 138, 476 Annotation: Subtype: Link Rect: 261, 443, 299, 453 Annotation: Subtype: Link Rect: 305, 443, 341, 453 Annotation: Subtype: Link Rect: 135, 409, 173, 419 Annotation: Subtype: Link Rect: 179, 409, 215, 419 Annotation: Subtype: Link Rect: 266, 374, 304, 384 Annotation: Subtype: Link Rect: 310, 374, 346, 384 Annotation: Subtype: Link Rect: 122, 340, 159, 350 Annotation: Subtype: Link Rect: 166, 340, 202, 350 Annotation: Subtype: Link Rect: 349, 316, 387, 327 Annotation: Subtype: Link Rect: 393, 316, 429, 327 Annotation: Subtype: Link Rect: 391, 293, 429, 304 Annotation: Subtype: Link Rect: 435, 293, 471, 304 Annotation: Subtype: Link Rect: 131, 259, 168, 269 Annotation: Subtype: Link Rect: 468, 235, 505, 246 Annotation: Subtype: Link Rect: 59, 202, 96, 212 Annotation: Subtype: Link Rect: 102, 202, 138, 212 Annotation: Subtype: Link Rect: 59, 179, 96, 189 Annotation: Subtype: Link Rect: 102, 179, 138, 189 Annotation: Subtype: Link Rect: 118, 144, 155, 154 Annotation: Subtype: Link Rect: 217, 121, 255, 131 Annotation: Subtype: Link Rect: 135, 87, 173, 97 Page: Sequence: 21 Annotations: Annotation: Subtype: Link Rect: 520, 710, 557, 721 Annotation: Subtype: Link Rect: 59, 699, 95, 710 Annotation: Subtype: Link Rect: 230, 676, 268, 687 Annotation: Subtype: Link Rect: 274, 676, 310, 687 Annotation: Subtype: Link Rect: 109, 653, 146, 664 Annotation: Subtype: Link Rect: 152, 653, 188, 664 Annotation: Subtype: Link Rect: 182, 630, 220, 640 Annotation: Subtype: Link Rect: 156, 607, 193, 617 Annotation: Subtype: Link Rect: 255, 583, 292, 594 Annotation: Subtype: Link Rect: 298, 583, 334, 594 Annotation: Subtype: Link Rect: 140, 561, 177, 571 Annotation: Subtype: Link Rect: 184, 561, 220, 571 Annotation: Subtype: Link Rect: 59, 527, 96, 537 Annotation: Subtype: Link Rect: 102, 527, 138, 537 Annotation: Subtype: Link Rect: 231, 503, 269, 514 Annotation: Subtype: Link Rect: 275, 503, 311, 514 Annotation: Subtype: Link Rect: 224, 481, 261, 491 Annotation: Subtype: Link Rect: 104, 458, 141, 468 Annotation: Subtype: Link Rect: 148, 458, 184, 468 Annotation: Subtype: Link Rect: 174, 435, 212, 445 Annotation: Subtype: Link Rect: 439, 411, 477, 422 Annotation: Subtype: Link Rect: 59, 377, 96, 387 Annotation: Subtype: Link Rect: 194, 343, 232, 353 Annotation: Subtype: Link Rect: 238, 343, 274, 353 Annotation: Subtype: Link Rect: 377, 319, 415, 330 Annotation: Subtype: Link Rect: 109, 285, 146, 295 Annotation: Subtype: Link Rect: 152, 285, 188, 295 Annotation: Subtype: Link Rect: 194, 216, 231, 226 Annotation: Subtype: Link Rect: 135, 193, 173, 203 Annotation: Subtype: Link Rect: 179, 193, 215, 203 Annotation: Subtype: Link Rect: 436, 169, 473, 180 Annotation: Subtype: Link Rect: 479, 169, 515, 180 Annotation: Subtype: Link Rect: 216, 136, 253, 146 Annotation: Subtype: Link Rect: 259, 136, 295, 146 Annotation: Subtype: Link Rect: 477, 112, 515, 123 Annotation: Subtype: Link Rect: 521, 112, 557, 123 Annotation: Subtype: Link Rect: 262, 77, 299, 88 Page: Sequence: 22 Annotations: Annotation: Subtype: Link Rect: 59, 711, 96, 721 Annotation: Subtype: Link Rect: 293, 687, 331, 698 Annotation: Subtype: Link Rect: 337, 687, 373, 698 Annotation: Subtype: Link Rect: 456, 664, 494, 675 Annotation: Subtype: Link Rect: 500, 664, 536, 675 Annotation: Subtype: Link Rect: 59, 630, 96, 640 Annotation: Subtype: Link Rect: 351, 606, 389, 617 Annotation: Subtype: Link Rect: 59, 573, 96, 583 Annotation: Subtype: Link Rect: 102, 573, 138, 583 Annotation: Subtype: Link Rect: 59, 538, 96, 548 Annotation: Subtype: Link Rect: 102, 538, 138, 548 Annotation: Subtype: Link Rect: 470, 514, 507, 525 Annotation: Subtype: Link Rect: 513, 514, 549, 525 Annotation: Subtype: Link Rect: 462, 491, 499, 502 Annotation: Subtype: Link Rect: 332, 468, 370, 479 Checksum: 23a46541 Type: CRC32 Checksum: 39c1ad849b7cf045b1ca419fbbb657cf Type: MD5 Checksum: 0dda89e9dd29dc1220ecaf15ac2f032f7434fdd9 Type: SHA-1