Ornithine Decarboxylase Knockdown Exacerbates Transient Focal Cerebral Ischemia-Induced Neuronal Damage in Rat Brain

Animals

Adult male, spontaneously hypertensive rats (SHR; 250 to 300 g; Charles River, Wilmington, MA, U.S.A.) were used in these studies. Rats were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals, U.S. Department of Health and Human Services Publication number 86–23 (revised 1986). All surgical procedures were approved by the Research Animal Resources and Care Committee of the University of Wisconsin-Madison.

Antisense knockdown

Ornithine decarboxylase antisense ODNs were designed based on rat ODC mRNA (Wen et al., 1989). Initial studies showed that the antisense ODN (5′-TCC TTA GTA AAG CTG CCC AT-3′) that targeted the translation initiation codon (nucleotides 434–453) was more effective than the antisense ODN (5′-TAA GAG CTA CAA GAA TGG CA-3′) that targeted the translation termination codon (nucleotides 1821–1840) in knocking-down ODC immunoreactive protein and catalytic activity. For routine studies, the translation initiation codon targeting antisense ODN was used. Control animals were infused with the sense (5-TCC TTA GTA AAG CTG CCC AT-3′) and the random (5′-TCA TTA CCG GGA TCC TCA AT-3′) ODNs, in which the proportion of each nucleotide is identical to that of the antisense ODN. None of the ODN sequences was related to any other nucleotide sequence in Genebank. The ODNs were synthesized with a phosphorothioate backbone and purified to analytical grade on high pressure liquid chromatography by Oligos Etc. (Wilsonville, OR, U.S.A.). The ODNs were infused as described earlier (Rothstein et al., 1996; Rao et al., 2001a, 2001b). The lyophilized ODNs were reconstituted (3.125 mg/mL) in artificial cerebrospinal fluid (aCSF; 119 mmol/L NaCl, 3.1 mmol/L KCl, 1.2 mmol/L CaCl₂, 1 mmol/L MgSO₄, 0.50 mmol/L KH₂ PO₄, 25 mmol/L NaHCO₃, 5 mmol/L D-glucose, 2.2 mmol/L urea, pH 7.4), dialyzed overnight (SpectraPor cellulose ester, MW cutoff 2,000; Thomas Scientific) at 4°C, filtered (0.22 μm), and filled into osmotic minipumps. Each pump was connected to a stainless steel cannula by peristaltic tubing and primed overnight at 37°C. The cannula was implanted into the lateral ventricle (coordinates −1.5 mm, lateral; −0.8 mm, anterior-posterior; −4.8 mm, dorsal-ventral; based on the rat brain atlas of Paxinos and Watson, 1998) and secured to the skull with dental cement. The pump was placed in the skin fold on the neck of the rat, under halothane anesthesia. The osmotic minipumps used were Alzet Model 2001 (Alza Pharmaceuticals, Palo Alto, CA, U.S.A.), which will pump at a rate of 1 μL/h for 7 days. Thus, each rat received 78 μg of ODN/day (3.125 μg/μL · hr). After 5 days of ODN infusion, rats were subjected to either transient middle cerebral artery occlusion (MCAO; 1 hour) or sham operation. After 1 day of reperfusion, rats were neurologically evaluated and perfused transcardially with buffered paraformaldehyde. The brains were then removed, postfixed and cryoprotected. The effect of antisense, sense, and random ODN infusion on the levels of ODC protein was evaluated by Western blotting. Proper functioning of the osmotic minipumps was confirmed by weighing the filled pumps before implantation and immediately after killing the rats. The average total volume of pumping was observed to be 136 ± 13 μL in 6 days (0.94 ± 0.09 μL/hr; n of 73). Correct placement of the cannula into the lateral ventricle was confirmed by examining the thionine stained brain slices.

Transient middle cerebral artery occlusion

Rats were anesthetized with halothane (induction: 2%; maintenance: 1.2%) in an oxygen and nitrous oxide (50:50) mixture. Rats were ventilated mechanically with a rodent ventilator (Model 683; Harvard Apparatus, South Natick, MA, U.S.A.) through an endotracheal tube (PE-240 polyethylene tubing; Harvard Apparatus). The left femoral artery was cannulated for continuous monitoring of arterial blood pressure and to obtain blood samples for the measurements of pH, PaO₂, PaCO₂, hemoglobin, and blood glucose concentration (I-STAT; Sensor Devices, Waukesha, WI, U.S.A.). PaO₂ and PaCO₂ were maintained between 100 to 200 mm Hg and 30 to 40 mm Hg, respectively.

Middle cerebral artery occlusion was conducted by an intraluminal suture technique as described previously (Zea Longa et al., 1989; Dogan et al., 1999; Rao et al., 2001b). The rat was placed in a stereotaxic frame, a craniectomy (4 mm in diameter, 2 to 4 mm lateral and 1 to 2 mm caudal to bregma) was performed with extreme care over the MCA territory using a trephine. The dura was left intact. A laser–Doppler flowmeter probe (model PD-434; Vasamedics, St. Paul, MN, U.S.A.) was placed on the surface of the ipsilateral cortex (ischemic area) and fixed to the periosteum with a 4–0 silk suture. The probe was connected to a laser flowmeter device (Laserflo blood perfusion monitor BPM 403A; TSI, St. Paul, MN, U.S.A.) for continuous monitoring of regional cerebral blood flow (rCBF). The left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were exposed through a ventral midline incision. A 3–0 monofilament nylon suture with a rounded tip was introduced into the ECA lumen and gently advanced to the ICA until slight resistance was felt and a reduction in rCBF was seen. The rCBF dropped to 12% to 18% of the baseline in 40 to 50 seconds and remained at that level throughout the occlusion period. After 1 hour of occlusion, the suture was withdrawn to restore the CCA-ICA-MCA blood flow (confirmed by laser–Doppler flowmeter). In less than 5 minutes after the withdrawal of the suture, the rCBF returned to the baseline level and remained unchanged through 90 minutes of reperfusion. Body and cranial temperatures were maintained with a heating blanket and a lamp at 37°C to 38°C and 36°C to 37°C, respectively, during the occlusion (1 hour) and 90 minutes of reperfusion. After recovering from anesthesia, rats were returned to their cages with free access to food and water. After one day, rats were perfused transcardially with buffered paraformaldehyde, and the brains were postfixed and cryoprotected.

Endpoint regional cerebral blood flow measurements

To confirm that ODC antisense ODN treatment had not changed the rCBF during ischemia, end ischemic rCBF was measured in additional cohorts by 4-iodo-[N-methyl- ¹⁴C]antipyrine ([¹⁴C]AIP) autoradiography as described earlier (Rusa et al., 1999; Rao et al., 2001b). For this, in a separate set of sense and antisense ODN-infused rats, the laser–Doppler flowmeter probes were attached, arterial and venous femoral catheters were inserted, and the MCA was occluded. At 60 minutes of occlusion, arterial blood pressure, PO₂, PCO₂, and pH were measured, and 40 μCi of [¹⁴C]AIP (specific activity, 54 mCi/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL, U.S.A.) in 0.8 mL of isotonic saline was infused intravenously for 45 seconds. Simultaneously, the arterial catheter was opened and 15 free-flowing 20-μL samples were collected into heparin-coated tubes. With the filament still in place and the laser–Doppler indicating the occlusion, the rat was decapitated and the brain was snap-frozen by dripping in isopentane cooled to −30°C and stored at −80°C. Each brain was sectioned (20-μm-thick) on a cryostat and the sections from 5 coronal levels (+2.2, +0.5, −0.9, −2.1, and −3.8 from Bregma) were collected and exposed for 1 week to hyperfilm βmax (Amersham) together with ¹⁴C standards. Blood samples were decolorized with tissue solubilizer and the radioactivity was estimated by liquid scintillation spectrometry. Autoradiographic images were digitized using MCID image analysis system (Imaging Research, St. Catherine's, Ontario, Canada) and the rates of rCBF were determined as previously described (Alkayed et al., 1998).

Histopathology

Each brain was sectioned coronally (40-μm-thick at an interval of 320 μm), stained with thionine, and scanned using the NIH Image program. The volume of the ischemic lesion was computed by the numeric integration of data from 16 to 19 serial sections in respect to sectional interval. To account for the cerebral edema and differential shrinkage resulting from tissue processing, the injury volumes were corrected using the following formula: corrected injury volume = contralateral hemisphere volume − (ipsilateral hemisphere volume − measured injury volume) (Swanson et al., 1990).

Neurologic evaluation

Neurologic deficits were evaluated on a 6-point scale (Zea Longa et al., 1989) before transient MCAO and at 1 day of reperfusion (before killing the animals) by an investigator blinded to the study groups. A score of 0 suggests no neurologic deficit (healthy); 1 suggests mild neurologic deficit (failure to extend right forepaw fully); 2 suggests moderate neurologic deficit (circling to the right), 3 suggests severe neurologic deficit (falling to the right), and 4 suggests very severe neurologic deficit (the rat did not walk spontaneously and had a depressed level of consciousness).

Western blotting

Immunoblotting was performed as described earlier (Rao et al., 1999). Brain tissue was homogenized in ice-cold 25 mmol/L Tris-HCl (pH 7.4) buffer containing 2 mmol/L EDTA and protease inhibitors (aprotinin, pepstatin-A, leupeptin, bestatin, 4-(2-aminoethyl) benzenesulfonyl fluoride, and trans-epoxysuccinyl-L-leucylamido(4-guanidino) butane). Protein content was estimated by the method of Lowry et al. (1951). Equal volume of Laemmli electrophoresis sample buffer (5% sodium dodecyl sulfate, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue and 0.125 mol/L Tris-HCl buffer, pH 6.8; Sigma, St. Louis, MO, U.S.A.) was added to 15 μg protein equivalent of each sample, and the proteins were solubilized by heating at 94°C for 3 minutes. Samples were electrophoresed on polyacrylamide gels, transferred to polyvinylidene difluoride, and probed with monoclonal anti-ODC antibody (Sigma) and horse radish peroxidase-coupled anti-mouse IgG. The blots were stripped and reprobed with anti-β-tubulin antibody (Sigma). The protein bands recognized by antibodies were visualized by enhanced chemiluminescence (ECL Western blotting kit; Amersham Pharmacia Biotech, Piscataway, NJ, U.S.A.) and the band intensities were quantified by densitometric scanning using the NIH Image program. Before immunodetection, blots were stained with Ponceau-S to confirm the protein loading and transfer efficiency.

Ornithine decarboxylase enzyme assay

Ornithine decarboxylase activity was assayed as described earlier (Rao et al., 1998). Brain tissue was homogenized in 50 mmol/L Tris-HCl buffer (pH 7.8) containing 40 μm pyridoxal-5-phosphate, 7.5 mmol/L dithiothreitol, 1 mmol/L ethylenediaminetetraacetic acid (EDTA), and 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged at 4°C (43,000 g for 20 minutes) and the supernatant was incubated in the buffer containing 0.5 μCi L-[¹⁴C]ornithine (specific activity, 47.7 Ci/mmol; NEN Life Sciences Products, Boston, MA, U.S.A.) at 37°C for 1 hour. Incubations were performed in airtight tubes with center wells containing filter paper strips soaked in sodium hydroxide. The ¹⁴CO₂ formed during the reaction trapped in the center wells was estimated by liquid scintillation spectrometry.

Statistical analysis

Results are presented as mean ± SD. Data from multiple groups were analyzed statistically by one-way analysis of variance followed by Tukey–Kramer multiple comparisons posttest. P < 0.05 was considered statistically significant.

Antisense infusion knocked down ornithine decarboxylase protein levels and activity

In sham-operated rats, cerebral cortex showed low ODC catalytic activity (5.8 ± 2.4 pmol/mg protein · h) and protein levels (Western blot analysis showed a faint ODC immunoreactive protein band similar to the contralateral cortex samples in Fig. 1). Transient MCAO induces ODC activity and protein levels in the ipsilateral cortex compared with the sham cortex, between 3 to 24 hours (with a peak at 6 hours) of reperfusion. Catalytic activity increased by 5-fold (at 3 hours), 9.9-fold (at 6 hours), and 4.7-fold (at 24 hours) in the ipsilateral cortex compared with the sham cortex. The ODC immunoreactive protein levels increased by 4.1-fold (at 3 hours), 7.4-fold (at 6 hours), and 3.7-fold (at 24 hours) in the ipsilateral cortex compared with the sham cortex. In comparison with the sham cortex, the contralateral cortex showed no significant changes in either the ODC activity or protein levels at any reperfusion period. Hence, in the current study, the efficiency of ODC antisense infusion on ODC protein knockdown was verified at 6 hours of reperfusion after transient MCAO. The ipsilateral cortex and striatum of the sense and MCAO group showed significantly greater ODC protein levels (by 5-to 6-fold, P < 0.01) compared with the respective contralateral cortex and striatum at 6 hours of reperfusion (Fig. 1). Infusion of ODC antisense significantly blocked the transient MCAO-induced increase in ODC protein level (by 84% ± 12%, P < 0.01) in ipsilateral cortex and striatum (Fig. 1). At 6 hours of reperfusion in the sense and MCAO group, there was a significant increase in the ODC catalytic activity in the ipsilateral cortex (by 3.5-fold, P < 0.01) and striatum (by 6.4-fold, P < 0.01), compared with the contralateral cortex and striatum (Fig. 2). Antisense infusion completely prevented the transient MCAO-induced increase in ODC activity in ipsilateral cortex and striatum (Fig. 2). Sham-operated rats infused with either sense or antisense showed little ODC activity (3.5 ± 0.6 and 2.6 ± 0.4 pmol/mg protein · h in cortex and striatum, respectively).

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FIG. 1.

Western blot analysis of ornithine decarboxylase (ODC) immunoreactive protein in the brains of rats undergoing transient middle cerebral artery occlusion (MCAO) after the infusion of sense and antisense oligonucleotides (ODNs). In the sense-infused rats, transient MCAO resulted in a significant increase in the ODC protein levels in the ipsilateral cortex and striatum compared with the contralateral cortex and striatum. Antisense infusion significantly prevented ischemia-induced increase in the ODC protein levels in the ipsilateral cortex and striatum. Cerebral cortex and striatum from the sham-operated controls showed little ODC protein levels bilaterally. Values in the histogram are mean ± SD of four animals in each group. For each animal, Western blotting was conducted twice. * P < 0.05 (sham vs. MCAO); †P < 0.05 (sense vs. antisense) by one-way analysis of variance followed by Tukey-Kramer multiple comparisons posttest. (top) Representative Western blots of ODC (top gel) and β-tubulin (bottom gel) immunoreactive protein levels in the ipsilateral cortex of antisense-and sense-infused rats, which underwent transient MCAO or sham-operation. Antisense treatment had no significant effect on the β-tubulin protein levels compared with the sense treatment.

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FIG. 2.

Antisense infusion significantly prevented the transient middle cerebral artery occlusion–induced increase in ornithine decarboxylase (ODC) activity. ODC activity was estimated in triplicate with <10% variation. Sham-operated rats infused with sense and antisense showed little ODC activity in cortex and striatum (3.5 ± 0.6 and 2.6 ± 0.4 pmol/mg protein/h, respectively). * P < 0.05 (contralateral vs. ipsilateral); †P < 0.05 (sense vs. antisense) by one-way analysis of variance followed by Tukey-Kramer multiple comparisons posttest.

Ornithine decarboxylase knockdown increased the ischemic infarct volume

In a group of rats not subjected to intracerebroventricular infusion, transient MCAO (1 hour) and reperfusion (24 hours) resulted in an infarct in the ipsilateral side of the brain with a total volume of 203 ± 28 mm³(cerebral cortex: 168 ± 31 mm³, striatum: 31 ± 5 mm³) (Table 1). Infusion of aCSF, sense, or random had no significant effect on the transient MCAO-induced infarct volume, whereas in the antisense/MCAO group, there was a significant increase in the total (by 33%;P < 0.05), cortical (by 35%, P < 0.05), and striatal (by 32%, P < 0.05) infarct volume compared with sense/MCAO or random/MCAO controls (Table 1). Figure 3 shows representative thionine-stained coronal sections from the brains of sense/MCAO, antisense/MCAO, sense/sham, and antisense/sham groups. There were no significant differences between the antisense/MCAO, sense/MCAO, and random/MCAO groups in any of the physiologic parameters (MABP, pH, PCO₂, PO₂, hemoglobin, and blood glucose) (Table 2) and the rCBF measured by laser–Doppler flowmetry (Fig. 4). The endpoint rCBF measured at 1 hour of MCAO, using [¹⁴C]AIP autoradiography, also was not significantly different between the sense and antisense-infused rats (Fig. 5).

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FIG. 3.

Thionine-stained serial coronal sections from the brains of representative rats infused with sense and antisense, which underwent transient middle cerebral artery occlusion (ischemia) or sham operation (sham). Figure shows only the sense-infused controls, as there were no observable differences between the sense-and random-infused groups. Mean ± SD of the total, cortical, and striatal infarct volume in each group is given in Table 1.

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FIG. 4.

Effect of sense (open circles) and antisense (filled circles) infusion on regional cerebral blood flow (rCBF) after transient middle cerebral artery occlusion (MCAO). Changes are expressed as percentage of the baseline. Values are mean ± SD. The rCBF was measured using a laser–Doppler flowmeter probe placed on the surface of the ipsilateral cortex (ischemic area) through a craniectomy over the MCA territory. There were no statistically significant differences between the two groups.

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FIG. 5.

Endpoint regional cerebral blood flow (rCBF) rates of 1 hour of middle cerebral artery occlusion as measured by autoradiography using [¹⁴C]AIP in the ornithine decarboxylase (ODC) sense- (n of 5) and antisense- (n of 5) infused rats. Flow rates in the representative areas of cerebral cortex and striatum were averaged across the contralateral and ipsilateral sides of the brain. No significant differences were observed between sense-and antisense-infused groups. The inset shows autoradiographs generated using the coronal brain sections (+2.2, +0.5, −0.9, −2.1, and −3.8 from Bregma) of [¹⁴C]AIP administered sense-and antisense-infused groups.

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TABLE 1.

Ischemic injury volumes in ODC sense, random, and antisense ODN-infused rats after transient MCAO

Intracerebroventricular infusion	n	Cortex	Striatum	Total
None	7	171 ± 31	31 ± 5	202 ± 36
aCSF	7	175 ± 36	34 ± 7	208 ± 39
Sense ODN	5	184 ± 37	37 ± 8	224 ± 53
Random ODN	5	186 ± 40	38 ± 9	224 ± 50
Antisense ODN	7	249 ± 56 * †	49 ± 8 * †	299 ± 63 * †

Data expressed in mm³. Values are mean ± SD. Middle cerebral artery occlusion (MCAO) was 1 hour in all cases. Ischemic injury volumes were measured at one day of reperfusion.

*

P < 0.05 compared with the none or artificial cerebrospinal fluid (aCSF) group

†

P < 0.05 compared with the sense oligonucleotides (ODN) or random ODN group (analysis of variance) followed by Tukey–Kramer multiple comparisons posttest).

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TABLE 2.

Physiologic parameters in ODC sense, random, and antisense ODN-infused rats before, during, and after MCAO

	Temperature (°C)		PaO2 (mm Hg)	PaCO2 (mm Hg)	pH	Hb (g/dL)	GI (mg/dL)	MABP
	Brain	Body
Preischemia
aCSF	36.7 ± 0.6	36.6 ± 0.6	14 ± 1.9	87 ± 7	7.36 ± 0.05	144 ± 29	33.6 ± 3.9	116 ± 11
Sense	36.3 ± 0.5	36.6 ± 0.4	14 ± 1.7	88 ± 9	7.42 ± 0.03	140 ± 33	34.5 ± 4.2	120 ± 10
Random	36.8 ± 0.6	36.9 ± 0.5	13 ± 1.8	91 ± 8	7.40 ± 0.06	137 ± 35	33.8 ± 4.7	118 ± 11
Antisense	36.7 ± 0.5	37.2 ± 0.6	14 ± 2.1	91 ± 7	7.39 ± 0.05	138 ± 30	34.7 ± 3.5	122 ± 9
During ischemia
aCSF	36.9 ± 0.5	37.2 ± 0.4	14 ± 1.4	88 ± 8	7.40 ± 0.06	137 ± 32	34.2 ± 4.5	115 ± 14
Sense	36.5 ± 0.4	36.9 ± 0.5	13 ± 1.7	91 ± 6	7.41 ± 0.05	136 ± 35	32.3 ± 4.7	118 ± 13
Random	36.5 ± 0.3	37.1 ± 0.6	13 ± 2.0	89 ± 8	7.43 ± 0.05	132 ± 28	32.9 ± 2.9	123 ± 10
Antisense	36.4 ± 0.4	36.7 ± 0.5	13 ± 1.9	90 ± 9	7.42 ± 0.04	142 ± 30	33.7 ± 3.3	120 ± 14
Postischemia
aCSF	36.4 ± 0.5	36.8 ± 0.6	13 ± 1.6	90 ± 9	7.37 ± 0.05	140 ± 36	33.6 ± 5.1	126 ± 12
Sense	36.9 ± 0.6	37.0 ± 0.4	14 ± 1.4	90 ± 8	7.44 ± 0.06	139 ± 34	34.1 ± 3.9	125 ± 13
Random	36.6 ± 0.5	37.3 ± 0.5	14 ± 2.2	91 ± 9	7.41 ± 0.04	137 ± 35	34.1 ± 3.9	120 ± 14
Antisense	36.7 ± 0.6	37.1 ± 0.6	14 ± 1.3	92 ± 8	7.38 ± 0.05	141 ± 34	32.8 ± 4.5	126 ± 13

HB, hemoglobin; GI, glucose; MAPB, mean arterial blood pressure; aCSF, artificial cerebrospinal fluid. Values are mean ± SD. There were no statistically significant differences among the different treatment groups (aCSF, sense, random, and antisense infused) at a specific period or within a treatment group at different periods (preischemia, during ischemia, and postischemia) in any of the parameters.

Ornithine decarboxylase knockout increased the transient MCAO-induced mortality and motor deficits

Transient MCAO (1 hour) resulted in 14% mortality and mild-to-moderate neurologic deficit at 24 hours of reperfusion in the rats not subjected to intracerebroventricular infusion (Table 3). There was no significant change in the mortality or neurologic deficit in the aCSF/MCAO, sense/MCAO, and random/MCAO groups (Table 3). Whereas, the antisense/MCAO group showed a significant increase in the mortality rate to 58% (P < 0.05) with a severe to very severe neurologic deficit (Table 3). There was no mortality or observable neurologic deficit in any of the sham groups (aCSF/sham, sense/sham, random/sham, and antisense/sham) (Table 3).

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TABLE 3.

ODC knockdown worsened the MCAO-induced neurologic deficit and mortality

	Median neuroscore (n × individual)	Neurologic deficit	Total rats (alive/dead)	% mortality
Sham
No infusion	0 (8 × 0)	None	8 (8/0)	0
aCSF	0 (8 × 0)	None	8 (8/0)	0
Sense ODN	0 (7 × 0)	None	7 (7/0)	0
Random ODN	0.14 (6 × 0, 1 × 1)	None	7 (7/0)	0
Antisense ODN	0.28 (5 × 0, 2 × 1)	None	7 (7/0)	0
MCAO
No infusion	2.14 * (6 × 2, 1 × 3)	Mild-moderate	8 (7/1)	13
aCSF	2.29 * (5 × 2, 2 × 3)	Mild-moderate	8 (7/1)	13
Sense ODN	2.09 * (7 × 2, 3 × 3)	Mild-moderate	10 (9/1)	9
Random ODN	2.20 * (4 × 2, 1 × 3)	Mild-moderate	6 (5/1)	17
Antisense ODN	3.64 * † (4 × 3, 7 × 4)	Severe-very severe	24 (11/13)	54 †

Middle cerebral artery occlusion (mCAO) was 1 hour. Neurologic evaluation was con-ducted at 24 hours of reperfusion, and neuroscores were not given for the rats that failed to survive up to this period. The scale for neurologic scoring is as follows: 0, no neurologic deficit; 1, mild neurologic deficit; 2, moderate neurologic deficit; 3, severe neurologic deficit; 4, very severe neurologic deficit.

*

P < 0.05 compared with the corresponding sham control

†

P < 0.05 compared with the respective sense/MCAO or random/MCAO control (analysis of variance followed by Tukey–Kramer multiple comparisons posttest). As 3 sham rats (1 random ODN-infused and 2 antisense ODN-infused) were scored at 1, 6 more sham rats (2 each infused with random, sense, and antisense ODNs) were run and all of them were scored as 0. Data for the 3 sham groups (random ODN, sense ODN, and antisense ODN) were consolidated from the 2 studies conducted 6 months apart.