Background: The initial 24 h after thrombolysis are critical for patients’ conditions, and continuous neurological assessment and blood pressure measurement are required during this time. The goal of this study was to identify the clinical factors associated with early neurological deterioration (END) within 24 h of stroke patients receiving intravenous recombinant tissue plasminogen activator (rt-PA) therapy and to clarify the effect of END on 3-month outcomes. Methods: A retrospective, multicenter, observational study was conducted in 10 stroke centers in Japan. A total of 566 consecutive stroke patients [211 women, 72 ± 12 years old, the median initial NIH Stroke Scale (NIHSS) score of 13] treated with intravenous rt-PA (0.6 mg/kg alteplase) was studied. END was defined as a 4-point or greater increase in the NIHSS score at 24 h from the NIHSS score just before thrombolysis. Results: END was present in 56 patients (9.9%, 18 women, 72 ± 10 years old) and was independently associated with higher blood glucose [odds ratio (OR) 1.17, 95% confidence intervals (CI) 1.07–1.28 per 1 mmol/l increase, p < 0.001], lower initial NIHSS score (OR 0.92, 95% CI 0.87–0.97 per 1-point increase, p = 0.002), and internal carotid artery (ICA) occlusion (OR 5.36, 95% CI 2.60–11.09, p < 0.001) on multivariate analysis. Symptomatic intracranial hemorrhage within the initial 36 h from thrombolysis was more common in patients with END than in the other patients (per NINDS/Cochrane protocol, OR 10.75, 95% CI 4.33–26.85, p < 0.001, and per SITS-MOST protocol, OR 12.90, 95% CI 2.76–67.41, p = 0.002). At 3 months, no patients with END had a modified Rankin Scale (mRS) score of 0–1. END was independently associated with death and dependency (mRS 3–6, OR 20.44, 95% CI 6.96–76.93, p < 0.001), as well as death (OR 19.43, 95% CI 7.75–51.44, p < 0.001), at 3 months. Conclusions: Hyperglycemia, lower baseline NIHSS score, and ICA occlusion were independently associated with END after rt-PA therapy. END was independently associated with poor 3-month stroke outcome after rt-PA therapy.

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