Neuropathological Characterization of Alzheimer's Disease

Medial temporal lobe (hippocampal and parahippocampal regions) samples were obtained from the tissue bank for neurological research Fundación CIEN (BT-CIEN; Centro de Investigación de Enfermedades Neurologicas; Madrid, Spain), the Neurological Tissue Bank of IDIBELL-Hospital of Bellvitge (Barcelona, Spain), and the Neurological Tissue Bank BioBanco-Hospital clínico-IDIBAPS (Barcelona, Spain). This study was approved by the Portal de Ética de la Investigación Biomédica de Andalucía (PEIBA) de la Consejería de Salud from Andalucía (Spain), as well as by the corresponding biobank ethics committees and by the “Comite de Etica de la Investigacion (CEI), Hospital Virgen del Rocio,” Seville, Spain. Subjects were classified according to Braak stage. Demographic and pathological characteristics of these cases are described in Table 1. Only Braak V–VI individuals (77.98 ± 12.48 years) met the clinical and neuropathological criteria for AD. Braak II subjects (77.50 ± 8.81) are considered as non-demented age-matched controls in this study. Younger subjects (49.50 ± 5.95 years) without any known psychiatric or neurological disease, and lack of neuropathological lesions, are referred as Braak 0.Table 1

Summary of the neuropathological samples used in this study

Braak stage	Age (years)	Gender (%)		APOE genotype (%)				Postmortem delay (h)
		Male	Female	ε2ε3	ε3ε3	ε3ε4	ε4ε4
Unfixed frozen sampes
Braak 0 (n = 8)	49.50 ± 5.95	62.50	37.50	0.00	66.67	0.00	33.33	7.19 ± 3.29
Braak II (n = 26)	77.50 ± 8.81	53.85	46.15	9.09	77.27	9.09	4.55	8.33 ± 5.05
Braak III–IV (n = 15)	79.42 ± 12.37	46.66	53.33	7.69	61.54	30.77	0.00	5.42 ± 4.80
Braak V–VI (n = 44)	77.98 ± 12.48	43.18	56.82	5.56	69.44	22.22	2.78	8.99 ± 4.55
Fixed samples
Braak 0 (n = 1)	58	100.00	0.00	–	–	–	––	5.00
Braak II (n = 8)	80.86 ± 6.77	28.57	71.43	–	–	–	–	10.67 ± 5.46
Braak V–VI (n = 14)	72.93 ± 14.06	64.29	35.71	14.29	28.57	57.14	0.00	7.65 ± 3.14

For biochemical characterization, unfixed −80 °C frozen samples were used. For morphological studies, human samples were fixed in cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 24–48 h, cryoprotected in sucrose, stored at −80 °C, sectioned at 30 μm thickness on a freezing microtome, and serially collected in wells containing 0.1 M phosphate buffer saline (PBS) and 0.02% sodium azide. The anatomical boundaries of the hippocampal and parahippocampal regions were identified in Nissl-stained sections by their cytoarchitecture and location using the human brain atlas [16 (link)]. The areas located between the stereotaxic coordinates of Bregma 9.3 mm and 34.6 mm were analyzed.