ACTA NEUROCHIRURGICA 9 by Springer-Verlag1979
Acta Neurochirurgiea 49, 207--217 (1979)
University Clinic of Neurosurgery, Graz (Head: Prof. Dr. F. Heppner) and Institute of Medical Biochemistry, Graz (Head: Prof. Dr. A. Holasek), Austria
Proteolytic Enzyme Activity in Patients With Severe Head Injury and the Effect of a Proteinase Inhibitor By
L. M. Auer, E. Marth, F. Heppner, and A. Holasek With I Figure Dedicated to Professor Dr. F. Spath on occasion of his 80th birthday
Summary A study was performed to detect the inhibitory effect of intravenously administered aprotinin (Trasylol | on brain and CSF protease activity in 25 patients with severe head injury. The data presented include measurements of CSF protease activity, alpha-l-antitrypsin, alpha-2-macroglobulin, haptoglobulin, polyacrylamidgel-electrophoresis pattern, total protein and hemoglobin content. The results indicate that increased protease activity is present and that this induces autolytic processes which can be inhibited by aprotinin treatment. The survival rate was higher after aprotinin treatment. Total CSF protein content was significantly higher in nonsurvivors than in survivors. Keywords: CSF-proteases; Head injury; Protease inhibitor.
Introduction In earlier reports e, 3 the possible role of proteolytic enzymes in injured brain tissue was discussed. In these we used experimental data f r o m a standardized head injury cat model. It was assumed that acid, and above all neutral proteases, are released f r o m damaged lysosomes within brain cells after head injury; the result of such an event can be further subcellular damage to mitochondria 22 and m e m brane structures within "autolysosomes", also called "cytolysosomes" and "autophagic vacuoles" "e, expressions indicating the autolytic nature of this process, which can include the cell membrane and lead to irreversible blood-brain barrier (BBB) dysfunction and spreading 0001-6268/79/0049/0207/$ 02.20
208
L.M. Auer et al.:
o f e n z y m e s into s u r r o u n d i n g structures. I n fact, initial clinical investigations s h o w e d t h a t p r o t e o l y t i c e n z y m e a c t i v i t y m a y be present in the cerebrospinal fluid (CSF) of patients w i t h severe head injury 4, possibly originating f r o m injured brain tissue. Interest thus developed in the question o f w h e t h e r such protease can be successfully inhibited in patients, a n d also w h e t h e r there are significant influences to be observed regarding clinical course and other biochemical values.
Selection of Patients and M e t h o d s Twenty-five patients were investigated. Each had suffered a severe head injury and remained comatose for at least seven days. Most of them presented with clinical brain stem signs (compression due to brain oedema or focal damage). Neurological diagnosis was supported by carotid angiography or computerized tomography, or both. In all patients, an external ventricular drain was implanted18, so. In 13 cases craniotomy was performed for evacuation of a haematoma or bitemporal decompression 13. All patients were treated in the same intensive care unit, and received parenteral nutrition via cava catheter, dexamethason regularly, mannitol and artificial ventilation if necessary. Twenty of these patients were included in a double blind study; nine patients were treated with the polyvalent protease inhibitor aprotinin (Trasylol | Bayer), (initially 500,000 IE, thereafter 200,000IE every four hours, intravenously). Eleven received a placebo drug. Five additional patients were added to the study and received aprotinin treatment. Biochemical Evaluation
CSF samples from the ventricular drain were taken daily for estimation of proteolytic enzyme activity by a spectral-photometrical technique with TAME and BAPA as substrates. CSF polyacrylamidgel-electrophoresis (PAGE) was performed for semiquantitative evaluation of pathological CSF protein patterns (Fig. 1). Therefore 10~ polyacrylamidgel in 8-molar urea was used, thus excluding molecules above a molecular weight of 200,000 from entering the gel. Three main areas of protein layers were distinguished (G I, II, III) (Fig. 1), and identified with numbers 1-3 according to the degree and intensity of occurrence and their sharp or blurred distinction from adjacent areas. The quantity of protein applied was equal in all CSF samples. Band G III was stained with benzidine, thus identifying haemoglobin of type A II (see Fig. 1). Total CSF protein content was estimated by Folin's method 1~. CSF haemoglobin was measured with a Merck kit. CSF alpha-l-antitrypsin and alpha-2-macroglobulin were measured by the immunodiffusion method. The usual routine examinations were mad8 from daily blood samples (electrolytes, urea, uric acid, creatinin, coagulatory factors, blood cells, Hb, HbE, osmolarity).
Results T h e lethal o u t c o m e in the whole series was 44O/o; four patients died in the a p r o t i n i n group, a n d six in the placebo group. F r o m the 56~ survivors, 21~ remained in an apallic state; four of them were treated w i t h aprotinin, one w i t h the placebo. O f the remaining
Proteolytic Enzyme Activity in Patients With Head Injury
209
survivors, six were in the aprotinin group and three in the placebo group. CSF protease activity was found in six cases, four of whom had been treated with aprotinin (for individual results see Tables 2 and 3). It was very low in all but one patient, mostly below 30 IU. In one patient with a value initially above 100 IU the protease activity was significantly regressive within the first week. Positive results on 11
Fig. 1. Typical CSF protein-PAGE pattern of a surviving patient during the first week after head injury and during aprotinin therapy. The intensity of three protein-band groups (G I, 1I, II1) was evaluated semiquantitatively: low intensity = 1, middle = 2, high = 3. The numbers on the picture are given in the respective order of G I-III. The day after injury is indicated underneath every single picture. Gel 0 from the day of injury shows denatured CSF proteins in bands G I and G II, which separate--on the second and third gel--into several well-defined bands. The third gel from day 4 after injury is already very similar to normal. Band G III indicates initially pathological haemoglobin A~, split into three layers by proteolytic activity, normalizing within the observation period
out of 200 occasions did not show a direct influence of aprotinin on CSF protease activity. However, several indirect observations suggested the presence of proteolytic activity, such as CS-PAGE (Fig. 1). The results of the individual CSF-PAGE patterns are given in Table 1. They show normalization of initially pathological protein 14 ActaNeurochirurgica,Vol. 49, Fasc. 3--4
210
L.M. Auer
et al.:
patterns in all nine aprotinin-treated cases within the first week of trauma. In the placebo group, this disappearance of the pathological protein pattern occurred in only 4 of 11 cases. Table 1.
Semiquantitative Evaluation of Polyacrylamid-gel Electrophoresis P a t i e n t D a y after h e a d injury Nr. 0 1 2 3 4 5
6
7
5
Trasylol 12 47 46 1 20 68 83 85 78 35 58 67 41 57
4 9 9
9
6 9 6 9 4 9 2 7 9
2 5 2
8
9
9
6
2 9 9 7 9 9 5 9 4 9
2 3
4 9 2 5
5
6
9
2 4
1
4 8 2 4
6 2
3
4
3 4
8
6
5
6 3 3
3 3
3 6
4 6
5 1 8
5
5 9 9
5
3 9 5 9 2 4
5 9 3 6
3
I
Placebo 81 13 14 23 62 63 65 50 64 77 61
7 2
9
5 9 7 9 3 9
9
6 9
3 6
5
9
4 4 9
2 4
CSF alpha-1 antitrypsin was investigated in four patients and found to be present in all of them. The values ranged between 5 and 67mg~ Alpha-2-macroglobulin was estimated in all patients and found to be positive in eight patients (see Table 2). Both CSF alpha-2-M and protease activity were found in three cases; one of the two substances occurred in seven patients. Protease activity was thus shown in ten cases. CSF haptoglobin was absent in all cases. Comparative examination of CSF Hb and blood Hb with PAGE
Proteolytic E n z y m e A c t i v i t y in Patients W i t h H e a d I n j u r y
211
showed marked splitting of CSF haemoglobin into several bands*. Single values of CSF total protein and haemoglobin are given in Table 3. Table 2. Patient Nr.
Alpha-2-Mmg~ (Proteases, I.U.)
D a y after head injury 0 i 2
3
4
5
6
7
0
0
0
0
0
0
0
0
0
0
0
0
2.77
0
0
Trasylol 12 47 46 1 20 68 83 85 78 35 58 67 41
-0 0 0 19 0 0 91 (2.43) 0 3.89 2.23 0 91
-0 0
20
0 176
0 0 0
0 --
0 --
0
75 1.8 (111.8)
0 0
0
0
0
0
0
0
0 234
0 0
0 0
0
0
I69
0 0 0
0 0
0 0 0
0
0
0 0 0 0
0 0 0 (18.5)
0 0 0 (16.6)
0
0 0 (17.2)
57
0.2 -(9.44)
0
0
0 65 5.95
Placebo 81 13 14 23 62 63 65 50 64 77 61
0
0 0 (I8.5)
0
0
0
0 0 51
0 0
Their very wide variation renders statistical evaluation impossible. A longer series, however, showed that total CSF protein is significantly higher in non-survivors than in survivors, thus giving * Stained w i t h benzidine a n d o-tolidin like hbA~, and n o r m a l i z i n g during a p r o t i n i n - t h e r a p y like the other bands. 14"
57
41
67
35 58
78
85
83
68
2O
1
46
47
12
Trasylol
Patient Nr.
2,849.6 (1.838)
(o)
532.7 (0.588) 48
(o)
46.7
(0.3427
464.1
410 (0.1)
1,697.2 (0.275) 61.3 (0) 99 (0.682)
179 (0.3) 297 (0.07) 85.6 457.5 (0.254) 48.8 (0) 617.1 (0.974) 30.2 (0) 201.5 (0.261)
Day after head injury 0 1
96.5 (0.044) 253.7 (0.239) 43.4 (0) 55.8 (0)
98 (0.06) 102 (07 71
1,798.8 (1.0447 55.6 (0) 41
2
50 (O)
32.8 (0)
59.7 (0)
76.5 (0)
(o7 38
30.3
(0) 72
(o)
21
38.6 (07 487.8 (0.184) 63.1 (0.085)
(o)
77.2
65.4
(o)
83.9 (0)
(o)
45.7 94.3
5
54.1
(o)
60.9
(o)
109.7
117.9 (0.029)
(o)
108
4
43.2
120.8 (0.183) 31.7 (0)
311.3 (0.176)
3
Table 3. Total CSF Protein in mg/ml (Haemoglobin in g ~
(0) 24
30.9
11.8 (0) 619.9 (0.389)
129.3 (0.184)
39.4
460.5 (8.63)
6
(o)
33.8
(o7
32.0
(o)
45.6
248.8 (0)
7
>
to
Placebo
61
77
64
50
65
63
23 62
14
13
81
(o)
26.6
682.9 (0.559) 140.6 (0.092)
245.9 (0.302) 216 (0.147) 694.7 (3.824)
79.7
(o)
113.8 (0.746) 287.7 (0.2) 51.1 (0.985) 2.2
(o)
152.8 (0.073) 55.3
122.9 (0.515)
143.1 (0.283) 63.3
825.5 (0.2)
(o)
71.1 (0.099) 122.3
(o)
71.4
(o)
200 (0.338) 165.9 (0.956) 39.2
67.7
(o) 135.5
(o) 130.3
40.7
(o)
264.8 (0.088)
(o)
82.5
55.6
36.2
(o)
196.2 (0.302)
53.2 (0.038)
45.9
(o)
41.0 (0.274) 220.4
(o)
(o)
183.2
(o)
(o) 181.9 (0.515) 30.7
71.2
88.9
228.9
(o)
59.3
(o)
39.1
236.6 (0.132)
(o)
41.3
280.9 (0.588)
(o)
125.1
r
s
g."
~a e-t
.~.
>
N ,,
Acta Neurochirurgiea 49, 207--217 (1979)
University Clinic of Neurosurgery, Graz (Head: Prof. Dr. F. Heppner) and Institute of Medical Biochemistry, Graz (Head: Prof. Dr. A. Holasek), Austria
Proteolytic Enzyme Activity in Patients With Severe Head Injury and the Effect of a Proteinase Inhibitor By
L. M. Auer, E. Marth, F. Heppner, and A. Holasek With I Figure Dedicated to Professor Dr. F. Spath on occasion of his 80th birthday
Summary A study was performed to detect the inhibitory effect of intravenously administered aprotinin (Trasylol | on brain and CSF protease activity in 25 patients with severe head injury. The data presented include measurements of CSF protease activity, alpha-l-antitrypsin, alpha-2-macroglobulin, haptoglobulin, polyacrylamidgel-electrophoresis pattern, total protein and hemoglobin content. The results indicate that increased protease activity is present and that this induces autolytic processes which can be inhibited by aprotinin treatment. The survival rate was higher after aprotinin treatment. Total CSF protein content was significantly higher in nonsurvivors than in survivors. Keywords: CSF-proteases; Head injury; Protease inhibitor.
Introduction In earlier reports e, 3 the possible role of proteolytic enzymes in injured brain tissue was discussed. In these we used experimental data f r o m a standardized head injury cat model. It was assumed that acid, and above all neutral proteases, are released f r o m damaged lysosomes within brain cells after head injury; the result of such an event can be further subcellular damage to mitochondria 22 and m e m brane structures within "autolysosomes", also called "cytolysosomes" and "autophagic vacuoles" "e, expressions indicating the autolytic nature of this process, which can include the cell membrane and lead to irreversible blood-brain barrier (BBB) dysfunction and spreading 0001-6268/79/0049/0207/$ 02.20
208
L.M. Auer et al.:
o f e n z y m e s into s u r r o u n d i n g structures. I n fact, initial clinical investigations s h o w e d t h a t p r o t e o l y t i c e n z y m e a c t i v i t y m a y be present in the cerebrospinal fluid (CSF) of patients w i t h severe head injury 4, possibly originating f r o m injured brain tissue. Interest thus developed in the question o f w h e t h e r such protease can be successfully inhibited in patients, a n d also w h e t h e r there are significant influences to be observed regarding clinical course and other biochemical values.
Selection of Patients and M e t h o d s Twenty-five patients were investigated. Each had suffered a severe head injury and remained comatose for at least seven days. Most of them presented with clinical brain stem signs (compression due to brain oedema or focal damage). Neurological diagnosis was supported by carotid angiography or computerized tomography, or both. In all patients, an external ventricular drain was implanted18, so. In 13 cases craniotomy was performed for evacuation of a haematoma or bitemporal decompression 13. All patients were treated in the same intensive care unit, and received parenteral nutrition via cava catheter, dexamethason regularly, mannitol and artificial ventilation if necessary. Twenty of these patients were included in a double blind study; nine patients were treated with the polyvalent protease inhibitor aprotinin (Trasylol | Bayer), (initially 500,000 IE, thereafter 200,000IE every four hours, intravenously). Eleven received a placebo drug. Five additional patients were added to the study and received aprotinin treatment. Biochemical Evaluation
CSF samples from the ventricular drain were taken daily for estimation of proteolytic enzyme activity by a spectral-photometrical technique with TAME and BAPA as substrates. CSF polyacrylamidgel-electrophoresis (PAGE) was performed for semiquantitative evaluation of pathological CSF protein patterns (Fig. 1). Therefore 10~ polyacrylamidgel in 8-molar urea was used, thus excluding molecules above a molecular weight of 200,000 from entering the gel. Three main areas of protein layers were distinguished (G I, II, III) (Fig. 1), and identified with numbers 1-3 according to the degree and intensity of occurrence and their sharp or blurred distinction from adjacent areas. The quantity of protein applied was equal in all CSF samples. Band G III was stained with benzidine, thus identifying haemoglobin of type A II (see Fig. 1). Total CSF protein content was estimated by Folin's method 1~. CSF haemoglobin was measured with a Merck kit. CSF alpha-l-antitrypsin and alpha-2-macroglobulin were measured by the immunodiffusion method. The usual routine examinations were mad8 from daily blood samples (electrolytes, urea, uric acid, creatinin, coagulatory factors, blood cells, Hb, HbE, osmolarity).
Results T h e lethal o u t c o m e in the whole series was 44O/o; four patients died in the a p r o t i n i n group, a n d six in the placebo group. F r o m the 56~ survivors, 21~ remained in an apallic state; four of them were treated w i t h aprotinin, one w i t h the placebo. O f the remaining
Proteolytic Enzyme Activity in Patients With Head Injury
209
survivors, six were in the aprotinin group and three in the placebo group. CSF protease activity was found in six cases, four of whom had been treated with aprotinin (for individual results see Tables 2 and 3). It was very low in all but one patient, mostly below 30 IU. In one patient with a value initially above 100 IU the protease activity was significantly regressive within the first week. Positive results on 11
Fig. 1. Typical CSF protein-PAGE pattern of a surviving patient during the first week after head injury and during aprotinin therapy. The intensity of three protein-band groups (G I, 1I, II1) was evaluated semiquantitatively: low intensity = 1, middle = 2, high = 3. The numbers on the picture are given in the respective order of G I-III. The day after injury is indicated underneath every single picture. Gel 0 from the day of injury shows denatured CSF proteins in bands G I and G II, which separate--on the second and third gel--into several well-defined bands. The third gel from day 4 after injury is already very similar to normal. Band G III indicates initially pathological haemoglobin A~, split into three layers by proteolytic activity, normalizing within the observation period
out of 200 occasions did not show a direct influence of aprotinin on CSF protease activity. However, several indirect observations suggested the presence of proteolytic activity, such as CS-PAGE (Fig. 1). The results of the individual CSF-PAGE patterns are given in Table 1. They show normalization of initially pathological protein 14 ActaNeurochirurgica,Vol. 49, Fasc. 3--4
210
L.M. Auer
et al.:
patterns in all nine aprotinin-treated cases within the first week of trauma. In the placebo group, this disappearance of the pathological protein pattern occurred in only 4 of 11 cases. Table 1.
Semiquantitative Evaluation of Polyacrylamid-gel Electrophoresis P a t i e n t D a y after h e a d injury Nr. 0 1 2 3 4 5
6
7
5
Trasylol 12 47 46 1 20 68 83 85 78 35 58 67 41 57
4 9 9
9
6 9 6 9 4 9 2 7 9
2 5 2
8
9
9
6
2 9 9 7 9 9 5 9 4 9
2 3
4 9 2 5
5
6
9
2 4
1
4 8 2 4
6 2
3
4
3 4
8
6
5
6 3 3
3 3
3 6
4 6
5 1 8
5
5 9 9
5
3 9 5 9 2 4
5 9 3 6
3
I
Placebo 81 13 14 23 62 63 65 50 64 77 61
7 2
9
5 9 7 9 3 9
9
6 9
3 6
5
9
4 4 9
2 4
CSF alpha-1 antitrypsin was investigated in four patients and found to be present in all of them. The values ranged between 5 and 67mg~ Alpha-2-macroglobulin was estimated in all patients and found to be positive in eight patients (see Table 2). Both CSF alpha-2-M and protease activity were found in three cases; one of the two substances occurred in seven patients. Protease activity was thus shown in ten cases. CSF haptoglobin was absent in all cases. Comparative examination of CSF Hb and blood Hb with PAGE
Proteolytic E n z y m e A c t i v i t y in Patients W i t h H e a d I n j u r y
211
showed marked splitting of CSF haemoglobin into several bands*. Single values of CSF total protein and haemoglobin are given in Table 3. Table 2. Patient Nr.
Alpha-2-Mmg~ (Proteases, I.U.)
D a y after head injury 0 i 2
3
4
5
6
7
0
0
0
0
0
0
0
0
0
0
0
0
2.77
0
0
Trasylol 12 47 46 1 20 68 83 85 78 35 58 67 41
-0 0 0 19 0 0 91 (2.43) 0 3.89 2.23 0 91
-0 0
20
0 176
0 0 0
0 --
0 --
0
75 1.8 (111.8)
0 0
0
0
0
0
0
0
0 234
0 0
0 0
0
0
I69
0 0 0
0 0
0 0 0
0
0
0 0 0 0
0 0 0 (18.5)
0 0 0 (16.6)
0
0 0 (17.2)
57
0.2 -(9.44)
0
0
0 65 5.95
Placebo 81 13 14 23 62 63 65 50 64 77 61
0
0 0 (I8.5)
0
0
0
0 0 51
0 0
Their very wide variation renders statistical evaluation impossible. A longer series, however, showed that total CSF protein is significantly higher in non-survivors than in survivors, thus giving * Stained w i t h benzidine a n d o-tolidin like hbA~, and n o r m a l i z i n g during a p r o t i n i n - t h e r a p y like the other bands. 14"
57
41
67
35 58
78
85
83
68
2O
1
46
47
12
Trasylol
Patient Nr.
2,849.6 (1.838)
(o)
532.7 (0.588) 48
(o)
46.7
(0.3427
464.1
410 (0.1)
1,697.2 (0.275) 61.3 (0) 99 (0.682)
179 (0.3) 297 (0.07) 85.6 457.5 (0.254) 48.8 (0) 617.1 (0.974) 30.2 (0) 201.5 (0.261)
Day after head injury 0 1
96.5 (0.044) 253.7 (0.239) 43.4 (0) 55.8 (0)
98 (0.06) 102 (07 71
1,798.8 (1.0447 55.6 (0) 41
2
50 (O)
32.8 (0)
59.7 (0)
76.5 (0)
(o7 38
30.3
(0) 72
(o)
21
38.6 (07 487.8 (0.184) 63.1 (0.085)
(o)
77.2
65.4
(o)
83.9 (0)
(o)
45.7 94.3
5
54.1
(o)
60.9
(o)
109.7
117.9 (0.029)
(o)
108
4
43.2
120.8 (0.183) 31.7 (0)
311.3 (0.176)
3
Table 3. Total CSF Protein in mg/ml (Haemoglobin in g ~
(0) 24
30.9
11.8 (0) 619.9 (0.389)
129.3 (0.184)
39.4
460.5 (8.63)
6
(o)
33.8
(o7
32.0
(o)
45.6
248.8 (0)
7
>
to
Placebo
61
77
64
50
65
63
23 62
14
13
81
(o)
26.6
682.9 (0.559) 140.6 (0.092)
245.9 (0.302) 216 (0.147) 694.7 (3.824)
79.7
(o)
113.8 (0.746) 287.7 (0.2) 51.1 (0.985) 2.2
(o)
152.8 (0.073) 55.3
122.9 (0.515)
143.1 (0.283) 63.3
825.5 (0.2)
(o)
71.1 (0.099) 122.3
(o)
71.4
(o)
200 (0.338) 165.9 (0.956) 39.2
67.7
(o) 135.5
(o) 130.3
40.7
(o)
264.8 (0.088)
(o)
82.5
55.6
36.2
(o)
196.2 (0.302)
53.2 (0.038)
45.9
(o)
41.0 (0.274) 220.4
(o)
(o)
183.2
(o)
(o) 181.9 (0.515) 30.7
71.2
88.9
228.9
(o)
59.3
(o)
39.1
236.6 (0.132)
(o)
41.3
280.9 (0.588)
(o)
125.1
r
s
g."
~a e-t
.~.
>
N ,,
