HISTOLOGICAL MARKERS OF DEGENERATION AND REGENERATION OF THE HUMAN INTERVERTEBRAL DISK

PELETTI-FIGUEIRÓ, MANUELA; AGUIAR, ISRAEL SILVEIRA DE; PAESI, SUELEN; MACHADO, DENISE CANTARELLI; ECHEVERRIGARAY, SERGIO; ROESCH-ELY, MARIANA; FALAVIGNA, ASDRUBAL; HENRIQUES, JOÃO ANTONIO PÊGAS

doi:10.1590/S1808-185120171601170833

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ORIGINAL ARTICLE • Coluna/Columna 16 (1) • Jan-Mar 2017 • https://doi.org/10.1590/S1808-185120171601170833 copy

HISTOLOGICAL MARKERS OF DEGENERATION AND REGENERATION OF THE HUMAN INTERVERTEBRAL DISK

MARCADORES HISTOLÓGICOS DE DEGENERAÇÃO E REGENERAÇÃO DO DISCO INTERVERTEBRAL HUMANO

MARCADORES HISTOLÓGICOS DE LA DEGENERACIÓN Y REGENERACIÓN DEL DISCO INTERVERTEBRAL HUMANO

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

Objective:

To define histological scores for intervertebral disc degeneration that would enable the definition of morphological characteristics of disease, besides improving knowledge of the lumbar degenerative disc disease by means of immunohistochemical markers.

Methods:

Hematoxylin and Eosin, Alcian/PAS, Masson Trichrome and Safranin O/FCF staining was used on the intervertebral disc degeneration sections of patients with lumbar degenerative disc disease. The protein markers defined in immunohistochemistry were cell proliferation (Ki-67) and apoptosis (p53).

Results:

The study data enabled the determination of Safranin O/FCF stain as the most effective one for evaluating parameters such as area, diameter, and number of chondrocyte clusters. The importance of using stains in association, such as Safranin O/FCF, Masson Trichrome, Alcian/PAS and Hematoxylin and Eosin, was also determined, as they are complementary for the histopathological verification of intervertebral disc degeneration. By expressing proteins using the immunohistochemistry technique, it was possible to consider two stages of disc degeneration: cell proliferation with chondrocyte cluster formation, and induction of apoptosis.

Conclusion:

This study enabled the histological and immunohistochemical characterization to be determined for lumbar degenerative disc disease, and its degrees of evolution, by determining new disc degeneration scores.

Keywords:
Intervertebral disc; Histology; Immunohistochemistry; Cell proliferation; Apoptosis

Stain	Purpose	Score-histology
Hematoxylin and Eosin (H&E)	Number of chondrocyte clusters in the NP	0- Absence
		1-Up to 50%
		2-50% up to 80%
		3->80%
	Mean horizontal line of the chondrocyte clusters in the NP	0-Absence
		1-Up to 9µm
		2->39µm up to 59µm
		3->59µm up to 79µm
		4->79µm
	Area of chondrocyte clusters in the NP	0-Absence
		1-Up to 1500µm²
		>1500µm² up to 2500 µm²
		>2500 µm² up to 3500 µm²
		>3500 µm² up to 4500 µm²
		>4500 µm²
	Fissure in the AF	0-Absence
		1-Presence
Safranin O/FCF	Number of chondrocyte clusters in the NP.	0-Absence
		1-Up to 50%
		2-50% up to 80%
		3->80%
	Mean Horizontal Line of the chondrocyte clusters in the NP	0-Absence
		1- Up to 39µm
		2->39µm up to 59µm
		3->59µm up to 79µm
	Area of the chondrocyte clusters in the NP	0-Absence
		1-Up to 1500µm²
		2->1500µm²up to 2500 µm²
		3->2500 µm² up to 3500 µm²
		4->3500 µm²up to 4500 µm²
		5->4500 µm²
Alcian/PAS (pH 2.1)	Acid mucopolysaccharide deposit around the chondrocyte clusters in the NP	0-Absence
		1-Rare
		2-Average
		3-Abundant
Masson Trichrome	Misalignment of the Collagen fibers around the clusters in the NP	0-Absence
		1-Rare
		2-Average
		3-Abundant

Correlations Spearman-Rho
		Ages	Time of evolution	Force	Sensibility	Disc degeneration	Facet degeneration	Alcian/PAS	Trichrome Masson	Scores for average area1	Average area1	Scores average diameter1	Average diameter1	Scores for number of clusters 1
Ages	Rho	1.000	0.375	-0.093	-0.021	0.422*	0.543**	0.102	0.107	-0.107	-0.186	0.083	0.025	0.100
	Sign	.	0.078	0.672	0.926	0.045	0.007	0.643	0.628	0.628	0.395	0.707	0.911	0.650
Time of evolution	Rho	0.375	1.000	-0.045	0.000	0.650**	0.311	-0.167	-0.164	-0.122	-0.252	-0.105	-0.148	-0.151
	Sign	0.078	.	0.838	1.000	0.001	0.148	0.447	0.454	0.578	0.247	0.633	0.500	0.493
Force	Rho	-0.093	-0.045	1.000	0.422*	0.199	0.150	0.345	0.126	0.031	0.022	0.024	-0.015	0.080
	Sign	0.672	0.838	.	0.045	0.362	0.495	0.098	0.567	0.890	0.919	0.914	0.946	0.715
Sensibility	Rho	-0.021	0.000	0.422*	1.000	0.044	0.163	0.382	-0.028	-0.007	0.164	0.090	0.125	0.226
	Sign	0.926	1.000	0.045	.	0.843	0.458	0.072	0.900	0.976	0.454	0.682	0.570	0.299
Disc degeneration	Rho	0.422*	0.650**	0.199	0.044	1.000	0.451*	-0.067	-0.049	-0.179	-0.267	-0.277	-0.239	-0.084
	Sign	0.045	0.001	0.362	0.843	.	0.031	0.760	0.823	0.414	0.219	0.201	0.272	0.703
Facet degeneration	Rho	0.543**	0.311	0.150	0.163	0.451*	1.000	0.084	-0.031	-0.121	-0.191	-0.033	0.023	0.070
	Sign	0.007	0.148	0.495	0.458	0.031	.	0.702	0.899	0.582	0.382	0.880	0.918	0.752
Alcian/PAS	Rho	0.102	-0.167	0.354	0.382	-0.067	0.084	1.000	0.350	0.096	0.265	0.377	0.269	0.584**
	Sign	0.643	0.447	0.098	0.072	0.760	0.702	.	0.102	0.664	0.221	0.076	0.214	0.003
Masson Trichrome	Rho	0.107	-0.164	0.126	-0.028	-0.049	-0.031	0.350	1.000	0.368	0.416*	0.351	0.340	0.494*
	Sign	0.628	0.454	0.567	0.900	0.823	0.889	0.102	.	0.084	0.048	0.100	0.112	0.017
Scores for average area1	Rho	-0.107	-0.122	0.031	-0.007	-0.179	-0.121	0.096	0.368	1.000	0.880**	0.817**	0.779**	0.543**
	Sign	0.628	0.578	0.890	0.976	0.414	0.582	0.664	0.084	.	0.000	0.000	0.000	0.007
Average area1	Rho	-0.186	-0.252	0.022	0.164	-0.267	-0.191	0.265	0.416*	0.880**	1.000	0.752**	0.892**	0.477*
	Sign	0.395	0.247	0.919	0.454	0.219	0.382	0.221	0.048	0.000	.	0.000	0.000	0.021
Scores for average diameter1	Rho	0.083	-0.105	0.024	0.090	-0.277	-0.033	0.377	0.351	0.817**	0.752**	1.000	0.795**	0.670**
	Sign	0.707	0.633	0.914	0.682	0.201	0.880	0.076	0.100	0.000	0.000	.	0.000	0.000
Average diameter1	Rho	0.025	-0.148	-0.015	0.125	-0.239	0.023	0.269	0.340	0.779**	0.892**	0.795**	1.000	0.474*
	Sign	0.911	0.500	0.946	0.570	0.272	0.918	0.214	0.112	0.000	0.000	0.000	.	0.022
Scores for number of clusters 1	Rho	0.100	-0.151	0.080	0.226	-0.084	0.070	0.584**	0.494*	0.543**	0.477*	0.670**	0.474*	1.000
	Sign	0.650	0.493	0.715	0.299	0.703	0.752	0.003	0.017	0.007	0.021	0.000	0.022	.

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[1] Correspondence: Asdrubal Falavigna Laboratório de Terapia Celular, Universidade de Caxias do Sul, Rua Francisco Getúlio Vargas, 1130, Caxias do Sul, RS, Brazil. 95070-560. asdrubalmd@gmail.com